Sirna/nanoparticle formulations for treatment of middle-east respiratory syndrome coronaviral infection

ABSTRACT

The present invention relates to compositions and methods for siRNA therapeutics for prevention and treatment of Middle East Respiratory Syndrome Corona Virus (MERS-CoV) infections. The compositions include a pharmaceutical composition comprising siRNA cocktails that target viral genes and pharmaceutically acceptable polymeric nanoparticle carriers and liposomal nanoparticle carriers.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a U.S. national phase application of, and claims thebenefit of and priority to, International Patent Application No.PCT/US2016/050590, filed Sep. 7, 2016, which claims the benefit of andpriority to U.S. Provisional Patent Application No. 62/215,565, filedSep. 8, 2015. The disclosures of these applications are expresslyincorporated herein by reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing that has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 26, 2018, isnamed SIR-014_P001-US_SL.txt and is 251,342 bytes in size.

FIELD OF INVENTION

The present invention provides a pharmaceutical product composition ofmatter comprising siRNA sequences targeting genes or single-strandedviral RNAs of Middle-East

Respiratory Syndrome Corona Virus (MERS-CoV), and nanoparticle carriersystems such as Histidine-Lysine co-polymers (HKP), or Spermine-Liposomeconjugates (SLiC), or a lung tissue targeted moiety, such as a peptide,a nucleotide, a small molecule, and an antibody. The present inventionalso involves in methods of use for this pharmaceutical product,including formulations of siRNA/nanoparticle carrier, their processdevelopment and specific delivery routes and regimens. This inventionpresents a novel therapeutic agent for treatment of MERS-CoV infection.

BACKGROUND

MERS-CoV Virus Disease: Biology and Pathology

Middle East respiratory syndrome (MERS) is a highly lethal respiratorydisease caused by a novel single-stranded, positive-sense RNAbetacoronavirus, MERS-CoV. Dromedary camels, hosts for MERS-CoV, areimplicated in direct or indirect transmission to human beings, althoughthe exact mode of transmission is unknown. Recent studies support thatcamels serve as the primary source of the MERS-CoV infecting humans,while bats may be the ultimate reservoir of the virus. The virus wasfirst isolated from a patient who died from a severe respiratory illnessin June, 2012, in Jeddah, Saudi Arabia. As of May 31, 2015, 1180laboratory-confirmed cases (483 deaths; 40% mortality) have beenreported to WHO (Zumbla A. et al. 2015). The Centers for Disease Controland Prevention (CDC) has labelled it as a transmissible disease fromhuman-to-humans. (Jalal S. 2015). Although most cases of MERS haveoccurred in Saudi Arabia and the United Arab Emirates, cases have beenreported in Europe, the USA, and Asia in people who travelled from theMiddle East or their contacts. Clinical features of MERS range fromasymptomatic or mild disease to acute respiratory distress syndrome andmultiorgan failure, resulting in death, especially in individuals withunderlying comorbidities. No specific drug treatment exists for MERS andinfection prevention, and control measures are crucial to prevent spreadin health-care facilities (Zumbla A. Et al 2015). Clinical severity ofthe disease observed in humans may be explained the ability of MERS-CoVto replicate in the lower respiratory tract (de Wit E, et al. 2013) andis also related to MERS-CoV's ability to infect a broad range of cellswith dipeptidyl peptidase 4 receptor (DPP4) expression, evade the hostinnate immune response, and induce cytokine dysregulation (Chan J F,2015).

MERS-CoV is an enveloped single-stranded positive sense RNA virus with agenome of 30,119 nt. The genome structure of MERS-CoV is similar toother coronaviruses, with the 5′ two-thirds of the genome encoding thenon-structural proteins (NSPs) required for viral genome replication,the remaining 3′ third of the genome encoding the structural genes thatmake up the virion (spike, envelope, membrane, and nucleocapsidproteins), and four accessory genes interspersed within the structuralgene region. At the 5′ end of the genome, there is a leader sequence(67nt), which is followed by an untranslated region (UTR). At the 3′ endof the RNA genome there is another UTR, followed by a poly (A) sequenceof variable length. Transcription-regulatory sequences (TRS 5′ AACGAA3′) are found at the 3′ end of the leader sequence and at differentpositions upstream of genes in the genomic 3′ -proximal domain ofMERS-CoV. The MERS-CoV genome contains at least 10 predicted openreading frames (ORFs): ORF1a, ORF1b, S, 3, 4a, 4b, 5, E, M and N withsixteen predicted nonstructural proteins being encoded by ORF1a/b.Several unique group-specific ORFs that are not essential for virusreplication are encoded by MERS-CoV. The functions of thesegroup-specific ORFs are unknown; however, by analogy to othercoronaviruses, they may encode structural proteins or interferonantagonist genes (Totura A L, Baric R S, 2012). Open reading framesORF2, -6, -7 and-8a are translated from subgenomic mRNAs predicted toencode the four canonical structural genes: a 180/90-kDa spikeglycoprotein (S), a˜23-kDa membrane glycoprotein (M), a small envelopeprotein (E) and a˜50-kDa nucleocapsidprotein (N), respectively(Abdel-Moneim A S. 2014).

Similar to other RNA viruses, coronavirus replicate in the hostcytoplasm. The replication process is initiated by the viral particleafter binding with specific cellular receptors, known as S-proteinmediated binding. The receptor for MERS-CoV was recently identified asdipeptidyl peptidase 4 (DDP4, also known as CD26), a protein withdiverse functions in glucose homeostasis, T-cell activation,neurotransmitter function, and modulation of cardiac signaling. DPP4 isexpressed in a variety of cell types, including endothelial cells(kidney, lung, small intestine, spleen) hepatocytes, enterocytes,activated leukocytes, testes, prostate and cells of the renal glomeruliand proximal tubules. DPP4 recognition is mediated by thereceptor-binding domain (RBD, amino acids E367-Y606) (Pascal K, et al.2015). Following virus entry, the coronavirus genome, a positive sense,capped and polyadenylated RNA strand, is directly translated, resultingin the synthesis of coronavirus replicase gene-encoded NSPs. CoronavirusNSPs are translated as two large polyproteins harboring proteolyticenzymes, namely papain-like and chymotrypsin-like proteinases thatextensively process coronavirus polyproteins to liberate up to 16 NSPs(nsp 1-16) (Lundin A. et al. 2014). After entering into the cell, thevirus specially modulates the innate immune response, antigenpresentation, and mitogen-activated protein kinase.

Current Prophylaxis and Therapeutics

Although the emergence of highly pathogenic MERS-CoV highlights anurgent need for potent therapeutic and prophylactic agents, no approvedantiviral treatments for any human coronavirus infections are currentlyavailable. Supportive treatment with extracorporeal membrane oxygenationand dialysis is often required in patients with organ failure. Recently,tremendous efforts have been made in the search for an effectiveanti-MERS-CoV agent, and a number of antiviral agents have beenidentified. For example, some compounds with inhibitory activities inthe low micromolar range on MERS-CoV replication in cell cultures havebeen identified from the libraries of FDA-approved drugs. de Wilde A H.and colleges identified four compounds (chloroquine, chlorpromazine,loperamide, and lopinavir) inhibiting MERS-CoV replication in thelow-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 μM)(de Wilde A H et al. 2014).

Antivirals with potent in vitro activities also include neutralizingmonoclonal antibodies, antiviral peptides, interferons, mycophenolicacid. It was reported that rhesus macaques treated with a cocktail ofIFN-a2b with ribavirin, a nucleoside analog, exhibited reduced MERS-CoVreplication and an improved clinical outcome (Falzarano D, et al. 2013).Lu L. and colleges designed and synthesized a peptide (HR2P) derivedfrom the HR2 domain in the S2 subunit of the spike (S) protein of theMERS-CoV EMC/2012 strain. They found that HR2P could bind with the HR1domain to form a stable six-helix bundle and thus inhibit viral fusioncore formation and S protein-mediated cell-cell fusion. HR2P wasdemonstrated to potently inhibit infection by both pseudotyped and liveMERS-CoV in different cell lines. After modification of the HR2P peptideby introducing Glu (E) and Lys (K) residues at the i to i+4 or i to i+3arrangements, it was found that one of these HR2P analogous peptides,HR2P-M2, exhibited significantly improved stability, solubility andantiviral activity. HR2P-M2 peptide could potently inhibit infection bypseudoviruses expressing MERS-CoV S protein with or without mutation inthe HR1 region, suggesting that it could be effective against mostcurrently available MERS-CoV mutants. It was demonstrated that theHR2P-M2 peptide administered via the intranasal route could protectAd5-hDPP4-transduced mice from challenge by MERS-CoV strains with orwithout mutations in the HR1 region, indicating that this peptide couldbe used as a nasal spray to protect high-risk populations, includinghealthcare workers, MERS patients' family members, and those havingclose contacts with the patients, from MERS-CoV infection. Intranasalapplication of the peptide to MERS-CoV-infected patients may suppressviral replication in epithelial cells of the respiratory tract and thusreduce the release of virions, thereby preventing the spreading ofMERS-CoV to other people (Lu L. et al. 2015).

Another approach is passive administration of sera from convalescenthuman MERS patients or other animals to exposed or infected patients.The vast majority of camels in the Middle East have been infected withMERS-CoV, and some contain high titers of antibody to the virus. It wasshown that this antibody is protective if delivered eitherprophylactically or therapeutically to mice infected with MERS-CoV,indicating that this may be a useful intervention in infected patients(Zhao J, et al. 2015).

In April 2014, three studies conducted by separate laboratories aroundthe world reported the development of fully human neutralizing mAbsagainst MERS-CoV. All these mAbs target the RBD (receptor-bindingdomain) of the MERS-CoV S1 glycoprotein and they were identified fromnon-immune human antibody libraries. Among these antibodies, threehighly potent mAbs (m336, m337, m338) were identified from a very largephage-displayed antibody Fab library that was generated by using B cellsfrom the blood of 40 healthy donors. This library was panned againstrecombinant MERS-CoV RBD to enrich for high affinity binders. The threeidentified mAbs, all derived from the VH gene 1-69, which has been thesource of many other antiviral antibodies, exhibited exceptionallypotent activity and neutralized pseudotyped MERS-CoV with 50% inhibitoryconcentration (IC50), ranging from 0.005 to 0.017 mg/ml. The most potentmAb, m336, inhibited>90% MERS-CoV pseudovirus infection (IC90) inDPP4-expressing Huh-7 cells at a concentration of 0.039 mg/ml.Similarly, m336 showed the most potent live MERS-CoV neutralizingactivity in inhibiting the formation of MERS-CoV-induced CPE during liveMERS-CoV infection of permissive Vero E6 cells, with an IC50 of 0.07mg/ml.

In vivo studies have shown that this mAb is very effective in protectingMERS-CoV-susceptible animals from viral challenge (unpublished data),suggesting that the m336m mAb is a very promising drug candidate for theurgent treatment of MERS-CoV-infected patients (Tianlei Ying et al.2015). Lu L. et colleges performed in vitro studies demonstrating thatthe combination of HR2P-M2 peptide with m336 mAb exhibited a strongsynergistic effect against MERS-CoV infection (unpublished data). Thisobservation suggests that intranasal administration of HR2P-M2 peptidecombined with intravenous administration of m336 mAb may be a powerfulstrategy for treatment of MERS patients (Lu L. et al. 2015).

Jiang and colleges also identified two potent RBD-specific neutralizingmAbs, MERS-4 and MERS-27, by using a non-immune yeast-displayed scFvlibrary to screen against the recombinant MERS-CoV RBD. The most potentmAb, MERS-4, neutralized the pseudotyped MERS-CoV infection inDPP4-expressing Huh-7 cells with an IC50 of 0.056 mg/ml and inhibitedthe formation of MERS-CoV-induced CPE during live MERS-CoV infection ofpermissive Vero E6 cells with an IC50 of 0.5 mg/ml. Tang et collegesidentified neutralizing mAbs by using a non-immune phage-displayed scFvlibrary. The panning was performed by sequentially using MERS-CoVspike-containing paramagnetic proteoliposomes and MERS-CoV Sglycoprotein-expressing 293T cells as antigens. A panel of 7 anti-S1scFvs was identified and expressed in both scFv-Fc and IgG1 formats, andtheir neutralizing activity against pseudotyped MERS-CoV inDPP4-expressing 293T cells, as well as live MERS-CoV infection in Verocells, was measured. The most potent antibody, 3B11, neutralized liveMERS-CoV in the plaque reduction neutralization tests with an IC50 of1.83 mg/ml and 3.50 mg/ml in the scFv-Fc and IgG format, respectively(Tianlei Ying et al. 2015).

Fully Human Antibody and Humanized Mouse Model

Pascal K. and colleges used the VelocImmune platform (a mouse thatexpresses human antibody-variable heavy chains and κ light chains) togenerate a panel of fully human, noncompeting monoclonal antibodies thatbind to MERS-CoV S protein and inhibit entry into target cells. It wasshowed that two of these antibodies (REGN3051 and REGN3048) can potentlyneutralize pseudoparticles generated with all clinical MERS-CoV S RBDvariants isolated to date. Authors demonstrated that the fully humanVelocImmune antibodies neutralize infectious MERS-CoV significantly moreefficient than published monoclonals isolated using traditional methods.They also developed a novel humanized model for MERS-CoV infection. Theyreplaced the 79 kb of the mouse Dpp4 gene with 82 kb of its humanortholog. The resulting mice express fully human DPP4 under the controlof the mouse regulatory elements, to preserve the proper expressionregulation and protein tissue distribution and showed that theseantibodies can prevent and treat MERS-CoV infection in vivo (Pascal K Eet al. 2015).

Coronaviruses

Coronaviruses are enveloped viruses and their positive strand RNAgenome, the largest of all RNA viruses, encodes for as many as 16non-structural proteins (NSPs), 4 major structural proteins, and up to 8accessory proteins. Many of these proteins provide essential, frequentlyenzymatic, functions during the viral life cycle, such as coronavirusprotease or RNA-dependent RNA polymerase (RdRp) activities. For example,the spike (S) protein mediates binding of different HCoVs to theirspecific cellular receptors, an event associated with preferential virustropism for either ciliated or non-ciliated cells of the airwayepithelium. The S protein also mediates fusion between lipids of theviral envelope and the host cell plasma membrane or membranes ofendocytic vesicles to promote delivery of viral genomic RNA into thecytoplasm. Following virus entry, the coronavirus genome, a positivesense, capped and polyadenylated RNA strand, is directly translated,resulting in the synthesis of coronavirus replicase gene-encoded NSPs.Coronavirus NSPs are translated as two large polyproteins harboringproteolytic enzymes, namely papain-like and chymotrypsin-likeproteinases that extensively process coronavirus polyproteins toliberate up to 16 NSPs (nsp 1-16). These proteolytic functions areconsidered essential for coronavirus replication. Likewise, thecoronavirus RdRp activities, which reside in nsp8 and nsp12, areconsidered essential for coronavirus replication. Coronaviruses encodean array of RNA-processing enzymes. These include a helicase activitylinked to an NTPase activity in nsp13, a 3′-5′-exonuclease activitylinked to a N7-methyltransferase activity in nsp14, an endonucleaseactivity in nsp15, and a 2′-O-methyltransferase activity in nsp16.

Like all positive strand RNA viruses, coronaviruses synthesize viral RNAat organelle-like structures in order to compartmentalize this criticalstep of the viral life cycle to a specialized environment that isenriched in replicative viral and host-cell factors, and at the sametime protected from antiviral host defense mechanisms. There is now agrowing body of knowledge concerning the involvement, rearrangement andrequirement of cellular membranes for RNA synthesis of a. number ofpositive-strand RNA viruses, including coronaviruses. Three coronaviralNSPs, i.e., nsp3, nsp4, and nsp6 are thought to participate in formationof these sites for viral RNA synthesis. In particular, these proteinscontain multiple trans-membrane domains that are thought to anchor thecoronavirus replication complex through recruitment of intracellularmembranes to form a reticulovesicular network (RVN) of modified,frequently paired, membranes that includes convoluted membranes anddouble membrane vesicles (DVM) interconnected via the outer membranewith the rough ER.

Culture Systems

MERS-CoV can replicate in different mammalian cell lines. In humans, itcan replicate in the respiratory tract (lung adenocarcinoma cell lineA549, embryonic fibroblast cell line HFL and polarized airway epitheliumcell line Calu-3), kidney (embryonic kidney cell line; HEK), liver cells(hepatocellular carcinoma cell line; Huh-7), and the intestinal tract(colorectal adenocarcinoma cell line; Caco-2). MERS-CoV can also infectcell lines originating from primates, pigs, bats, civet cats and rabbits(Chan et al. 2013).

Additional Mouse Models

Zhao J and colleges described a novel approach to developing a mousemodel for MERS by transducing mice with a recombinant, nonreplicatingadenovirus expressing the hDPP4 receptor. After infection with MERS-CoV,mice develop an interstitial pneumonia. Similar to infected patients,Ad5-hDPP4-transduced mice with normal immune systems developed milddisease whereas immunocompromised mice, like patients with underlyingdiseases, were more profoundly affected. It was shown that thesetransduced, infected mice can be used to determine antivirus immuneresponses and to evaluate anti-MERS-CoV vaccines and therapies (Zhao J,et al. 2014).

Two Mouse Models have been developed Pascal K et al. In the first, amodified adenovirus expressing huDPP4 was administered intranasally tomice leading to huDPP4 expression in all cells of the lung, not justthose that natively express DPP4. In this model, mice showed transienthuDPP4 expression and mild lung disease. In the second model, atransgenic mouse was produced to expresses huDPP4 in all cells of thebody, which in not physiologically relevant. In this model, MERS-CoVinfection leads to high levels of viral RNA and inflammation in thelungs, and also significant inflammation and viral RNA in the brains ofinfected mice. However, no previous reports have documented tropism ofMERS-CoV to the brains of an infected host, suggesting that studyingpathogenesis of MERS-CoV in this model is limited.

RNAi and siRNA

RNA interference (RNAi) is a sequence-specific RNA degradation processthat provides a direct way to knockdown, or silence, theoretically anygene. In naturally occurring RNA interference, a double stranded RNA iscleaved by an RNase III/helicase protein, Dicer, into small interferingRNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-ntoverhangs at the 3′ ends. These siRNAs are incorporated into amulticomponent-ribonuclease called RNA-induced-silencing-complex (RISC).One strand of siRNA remains associated with RISC, and guides the complextowards a cognate RNA that has sequence complementary to the guiderss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA,thereby inactivating it. Studies have revealed that the use ofchemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammaliancells, and the thermodynamic stability of siRNA hybridization (atterminals or in the middle) plays a central role in determining themolecule's function.

Importantly, it is presently not possible to predict with high degree ofconfidence which of many possible candidate siRNA sequences potentiallytargeting an mRNA sequence of a disease gene will, in fact, exhibiteffective RNAi activity. Instead, individually specific candidate siRNApolynucleotide or oligonucleotide sequences must be generated and testedto determine whether the intended interference with expression of atargeted gene has occurred.

Target Selection

MERS-CoV is enveloped single-stranded positive-sense RNA viruses,belonging to genus Betacoronavirus. The length of the genome is around30 k nt. The genome contains 10 predicted open reading frames (ORFs):ORF1a, ORF1b, Spike (S) Protein, 3, 4a, 4b, 5, Envelope (E) Protein,Membrane (M) Protein and Nucleocapsid (N) Protein, with 5′ two third ofthe genome (ORF1a, ORF1b) encoding 16 non-structure proteins (nsp1-16),and rest 3′ third of the genome encoding 4 structure proteins (S, E, Mand N proteins).

The spike (S) protein of MERS-CoV is a glycoprotein with a molecularweight of 180/190 kDa, which is an important determinant of virusvirulence and host range. Trimers of S protein form the spikes on theMERS-CoV envelope, which are responsible for the receptor binding andmembrane fusion. Similar to the HIV envelope (env) and influenzahemagglutinin (HA), S proteins of MERS-CoV are Class I viral fusionproteins, which requires the protease cleavage between the S1 and S2domains to allow the conformational changes in S2, and initiate thevirus entry and syncytia formation. Dipeptidyl peptidase 4 (DDP4, orCD26), a protein with diverse functions in glucose homeostasis, T-cellactivation, etc., has been identified as the receptor of MERS-CoV on thehost cells. The recognition of DPP4 is mediated by the receptor-bindingdomain (RBD, aa E367-Y606) of the S protein. DPP4 is expressed in avariety of cell types. It has been discovered on the human cell surfacein the airways (such as the lungs) and kidneys recently.

After entry into the cell, two polyproteins, pp1a and pp1ab of MERS-CoVexpress and undergo cotranslational proteolytic processing into theproteins that form the viral replication complex. During thisprocessing, the activity of nsp-3, papain-like protease (PL^(pro)) andnsp-5, 3C-like proteinase (3CL^(pro)) are critical for the generation of16 nonstructural proteins from the polyprotein. However, based on theMERS-CoV genome sequences analysis and calculation, we found severalsiRNA candidates (MPL1-6) match PL^(pro) as the target, but no goodcandidate matches 3CL^(pro). Meanwhile, the recent studies showed thatMERS-CoV PL^(pro) also has the function to inhibit the innate immuneresponse to viral infection by decreasing the levels of ubiquitinatedand ISGylated host cell proteins and down-regulating the cytokines, suchas CCLS and IFN-β in stimulated cells.

MERS-CoV RNA-dependent RNA polymerase (RdRp), encoding by nsp-12, is themost important component of viral replication complex. This complex isresponsible for both the transcription of the nested subgenomic mRNAsand the replication of the genomic positive-strand RNA. Both processestake place in the cytoplasm. In the viral mRNA transcription, thenegative-strand RNAs generate from genomic RNA at first, and thentranscribe a set of 3′-coterminal nested subgenomic mRNAs by thereplication complex, with a common 5′ “leader” sequence (67nt) derivedfrom the 5′ end of the genome. The newly synthetic genomic RNAs areproduced by the taking the negative-strand RNAs as the template.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The genome structure of MERS-CoV MERS-CoV is envelopedsingle-stranded positive-sense RNA viruses, belonging to genusBetacoronavirus, with a genome of ˜30K nt. The genome contains 10predicted open reading frames (ORFs): ORF1a, ORF1b, Spike (S) Protein,3, 4a, 4b, 5, Envelope (E) Protein, Membrane (M) Protein andNucleocapsid (N) Protein with 16 predicted nonstructural proteins beingencoded by ORF1a/b.

FIG. 2. The life cycle of MERS. After binding to the receptor, viral RNAand proteins of MERS-CoV are synthesized entirely in the cytoplasm. Twopolyproteins, pp1a and pp1ab undergo cotranslational proteolyticprocessing into the proteins that form the viral replication complex.This complex is used to produce the negative-strand RNA from genomicRNA, and transcribe a 3′-coterminal set of nested subgenomic mRNAs fromthe negative-strand RNA, which have a common 5′ “leader” sequencederived from the 5′ end of the genome. This viral replication complex isalso used to produce the positive-strand genomic RNA taking thenegative-strand RNA as the template.

FIG. 3. Special design of siRNA sequences targeting critical viralgenes: Papain like protein (PL^(pro)) specific siRNA, total 6 activesiRNAs (MPL1-6); RNA dependent RNA protease (RDRP) specific siRNA, total5 active siRNAs (MRR1-5) and Spike protein specific siRNA, total 8active siRNAs (MSP1-8). FIG. 3 discloses SEQ ID NOS 1-2, 132, 4-12, 700,and 13-18, respectively, in order of columns.

FIG. 4. Histidine-Lysine co-polymer enhances topical and subcutaneoussiRNA deliveries in vivo. The self-assembled HKP/siRNA nanoparticles(average 150 nm in diameter) can be dissolved in aqueous solution, canbe lyophilized into dry powder, and can be redissovled and mixed withmethylcellulose, or with RNAse free water. HKP/siRNA nanoparticledelivery to mouse respiratory track: upper airway, bronchi, alveoli.

FIG. 5. Comparison of target knockdown of lung endogenous gene amongHKP, DOTAP and D5W after oral tracheal deliveries of siRNA with threedifferent dosing regimens. HKP demonstrated the efficient siRNA-mediatedknockdown of the target gene at the 20 μg dose.

FIG. 6. Intraperitoneal delivery of HKP-siRNA nanoparticle formulationdemonstrated a prophylactic effect against H1N1 in the viral challengedmice (n=10). The evidence of the anti-influenza efficacy achieved byHKP-siRNA respiratory delivery support our notion that the similarapproach can also be applied for anti-MERS siRNA therapeutics. TheHKP-siRNA combination (siRNA103-siRNA105 with a 1:1 ratio) at aconcentration of 40 μg/2m1 was intraperitoneally administrated on day 1,2, 3, 4 and 5 (2.5 mg/kg/day). The viral challenges through intranasaladministrations of 2x LD50 H1N1 (A/Puerto Rico/8/1934) were conducted onday 2 ( ) for the virus only, Ribavirin and siRNA treatment groups.Ribavirin as a positive control was administered through gavages of200u1 to provide 75 mg/kg/day dosing over days 1-5 ( ). The prophylacticefficacy of HKP-siRNA formulation is clearly better than that ofRibavirin.

FIG. 7. Intraperitoneal delivery of PAA-siRNA formulation demonstrated atherapeutic efficacy against H1N1 in the viral challenged mice (n=15).The viral challenges through intranasal administrations of 1x LD50 H1N1(A/California/04/2009) were conducted on day 1 ( ) for the virus only,Tamiflu® and siRNA treatment groups. The H1N1 challenged mice weretreated with various dosages of PAA-siRNA combination (siRNA89-siRNA103with a 1:1 ratio), 1 mg/kg, 5 mg/kg and 10 mg/kg, throughintraperitoneal administration daily, from day 2 to day 6 ( ). Adaptingthe same route and dosing regimen, 25 mg/kg Tamiflu® ID was alsoadministrated daily on the H1N1 infected mice. The therapeutic efficacyof 10 mg/kg/day of PAA-siRNA combination resulted in almost equalanti-influenza activity to that of 25 mg/kg/day of Tamiflu® treatment.

FIG. 8. Scheme of the Basic Synthesis Routes and Structure ofSpermine-Liposome Conjugates (SLiC) A. The synthesis route for each ofthe five molecules are listed with the specific liposome chain, such as,R₁, R₂, R₃, R₄ and R₅, conjugated at the location of R₁H, R₂H, R₃H, R₄Hand R₅H respectively. B. The structures of the five SLiC species areillustrated with a spermine head and two lipid legs.

FIG. 9. Target Gene Silencing by SLiC Liposome-Mediated siRNA DeliveryIn Vivo. TM4-packaged siRNA specific to cyclophilin-B was selected forbeing tested in a Balb/c mouse model through a respiratory route ofdelivery. In addition to Blank control and empty TM4 control, a HKPpackage cyclophilin-B siRNA was used as a positive control. Threedifferent dosage: 25, 40 and 50 μg were tested. Both 40 and 50 μg siRNAdosages achieved significant target gene silencing (N=3,*P<0.05).

FIG. 10. Evaluation of the cytokine response in the mouse lung afterHKP-siRNA nanoparticles delivery. HKP-siRNA at different dosages wereoraltracheally administrated in the mouse lungs. The total lung tissuewere harvested for protein isolation and cytokine measurements by ELISAassay.

FIG. 11. A. Standard curve to measure protein concentration was preparedaccording to in-house SOP (Lowry Method); B. Total protein concentrationwas determined in each sample.

FIG. 12. A. Standard curve to measure TNF-α concentration was preparedaccording to in-house SOP (Lowry Method); B. TNF-α concentration in eachsample was determined and normalized to total protein.

FIG. 13. A. Standard curve to measure IL-6 concentration was preparedaccording to in-house SOP (Lowry Method); B. IL-6 concentration in eachsample was determined and normalized to total protein.

FIG. 14. A. Standard curve to measure IFN-α concentration was preparedaccording to in-house SOP (Lowry Method); B. IFN-α concentration in eachsample was determined and normalized to total protein.

FIG. 15. The HKP siRNA nanoparticle aqueous solution and SLiC siRNAnanoparticle aqueous solution will be administrated through airway,using an ultrasound nebulizer generated aerosol which will have watersolution particle size with broad spectrum allowing whole lungdistribution.

DESCRIPTION OF THE INVENTION

The present invention provides siRNA molecules that inhibit MERS-CoVgene expression, compositions containing the molecules, and methods ofusing the molecules and compositions to prevent or treat MERS in asubject, such as a human patient.

SiRNA Molecules

As used herein, an “siRNA molecule” or an “siRNA duplex” is a duplexoligonucleotide, that is a short, double-stranded polynucleotide, thatinterferes with the expression of a gene in a cell, after the moleculeis introduced into the cell, or interferes with the expression of aviral gene. For example, it targets and binds to a complementarynucleotide sequence in a single stranded (ss) target RNA molecule. SiRNAmolecules are chemically synthesized or otherwise constructed bytechniques known to those skilled in the art. Such techniques aredescribed in U.S. Pat. Nos. 5,898,031, 6,107,094, 6,506,559, 7,056,704and in European Pat. Nos. 1214945 and 1230375, which are incorporatedherein by reference in their entireties. By convention in the field,when an siRNA molecule is identified by a particular nucleotidesequence, the sequence refers to the sense strand of the duplexmolecule.

One or more of the ribonucleotides comprising the molecule can bechemically modified by techniques known in the art. In addition to beingmodified at the level of one or more of its individual nucleotides, thebackbone of the oligonucleotide can be modified. Additionalmodifications include the use of small molecules (e.g. sugar molecules),amino acids, peptides, cholesterol, and other large molecules forconjugation onto the siRNA molecule.

The siRNA molecules of the invention target a conserved region of thegenome of a MERS-CoV. As used herein, “target” or “targets” means thatthe molecule binds to a complementary nucleotide sequence in a MERS-CoVgene, which is an RNA molecule, or it binds to mRNA produced by thegene. This inhibits or silences the expression of the viral gene and/orits replication. As used herein, a “conserved region” of a MERS-CoV geneis a nucleotide sequence that is found in more than one strain of thevirus, is identical among the strains, rarely mutates, and is criticalfor viral infection and/or replication and/or release from the infectedcell.

In one embodiment, the siRNA molecule is a double-strandedoligonucleotide with a length of about 17 to about 27 base pairs. In oneaspect of this embodiment, the molecule is a double-strandedoligonucleotide with a length of 19 to 25 base pairs. In another aspectof this embodiment, the molecule is a couple-stranded oligonucleotidewith a length of 19 to 25 base pairs. In still another aspect of thisembodiment, it is a double-stranded oligonucleotide with a length of 25base pairs. In all of these aspects, the molecule may have blunt ends atboth ends, or sticky ends with overhangs at both ends (unpaired basesextending beyond the main strand), or a blunt end at one end and asticky end at the other. In one particular aspect, it has blunt ends atboth ends. In another particular aspect, the molecule has a length of 25base pairs (25 mer) and has blunt ends at both ends.

In one embodiment, the conserved MERS-CoV genomic regions are the genesequences coding for the MERS-CoV proteins Papain-like protease(PL^(pro)), RNA-dependent RNA polymerase (RdRp), and Spike protein. Thegenomic locations of such genes are shown in FIG. 3. In one embodiment,the siRNA molecule targets PL^(pro) virus gene expression. In anotherembodiment, the siRNA molecule targets RdRp viral gene expression. Instill another embodiment, the siRNA molecule targets Spike viral geneexpression.

Particular siRNA sequences that represent some of the siRNA molecules ofthe invention are disclosed in Tables 1-3. In one embodiment, the siRNAmolecules are disclosed in Table 3. In one particular embodiment, thesiRNA molecules are the following:

MPL1: CGCAAUACGUAAAGCUAAAGAUUAU, (SEQ ID NO: 1)MPL2: GGGUGUUGAUUAUACUAAGAAGUUU, (SEQ ID NO: 2)MPL3: CGCAUAAUGGUGGUUACAAUUCUU, (SEQ ID NO: 3)MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU, (SEQ ID NO: 4)MPL5: GCGCUUUUACAAAUCUAGAUAAGUU, (SEQ ID NO: 5)MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU, (SEQ ID NO: 6)MRR1: CCCAGUGUUAUUGGUGUUUAUCAUA, (SEQ ID NO: 7)MRR2: GGGAUUUCAUGCUUAAAACAUUGUA, (SEQ ID NO: 8)MRR3: GGGUGCUAAUGGCAACAAGAUUGUU, (SEQ ID NO: 9)MRR4: CCCCAAAUUUGUUGAUAAAUACUAU, (SEQ ID NO: 10)MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU, (SEQ ID NO: 11)MSP1: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 12)MSP2: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 12)MSP3: CCGAAGAUGAGAUUUUAGAGUGGUU, (SEQ ID NO: 13)MSP4: CCCAGUUUAAUUAUAAACAGUCCUU, (SEQ ID NO: 14)MSP5: GGCUUCACUACAACUAAUGAAGCUU, (SEQ ID NO: 15)MSP6: CCCCUGUUAAUGGCUACUUUAUUAA, (SEQ ID NO: 16)MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, (SEQ ID NO: 17) andMSP8: GCCGCAUAAGGUUCAUGUUCACUAA. (SEQ ID NO: 18)

In one embodiment, the targeted conserved regions of the genome comprisegene sequences coding for the following MERS-CoV proteins: Papain-likeprotease (PL^(pro)), RNA-dependent RNA polymerase (RdRp), and Spikeprotein. In one aspect of this embodiment, the siRNA molecules targetPL^(pro) viral gene expression. Such siRNA molecules include thefollowing:

MPL1: CGCAAUACGUAAAGCUAAAGAUUAU, (SEQ ID NO: 1)MPL2: GGGUGUUGAUUAUACUAAGAAGUUU, (SEQ ID NO: 2)MPL3: CGCAUAAUGGUGGUUACAAUUCUU, (SEQ ID NO: 3)MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU, (SEQ ID NO: 4)MPL5: GCGCUUUUACAAAUCUAGAUAAGUU, (SEQ ID NO: 5) andMPL6: CGCAUUGCAUGCCGUAAGUGUAAUU. (SEQ ID NO: 6)

In another aspect of this embodiment, the siRNA molecules target RdRpviral gene expression. Such siRNA molecules include the following:

The siRNA molecules of the invention also include ones derived fromthose listed in Tables 1-3 and otherwise herein. The derived moleculescan have less than the 25 base pairs shown for each molecule, down to 17base pairs, so long as the “core” contiguous base pairs remain. That is,once given the specific sequences shown herein, a person skilled in theart can synthesize molecules that, in effect, “remove” one or more basepairs from either or both ends in any order, leaving the remainingcontiguous base pairs, creating shorter molecules that are 24, 23, 22,21, 20, 19, 18, or 17 base pairs in length, if starting with the 25 basepair molecule. For example, the derived molecules of the 25 mermolecules disclosed in Tables 1-3 include: a) 24 contiguous base pairsof any one or more of the molecules; b) 23 contiguous base pairs of anyone or more of the molecules; c) 22 contiguous base pairs of any one ormore of the molecules; b) 21 contiguous base pairs of any one or more ofthe molecules; d) 20 contiguous base pairs of any one or more of themolecules; e) 19 contiguous base pairs of any one or more of themolecules; f) 18 contiguous base pairs of any one or more of themolecules; and g) 17 contiguous base pairs of any one or more of themolecules. It is not expected that molecules shorter than 17 base pairswould have sufficient activity or sufficiently low off-target effects tobe pharmaceutically useful; however, if any such constructs did, theywould be equivalents within the scope of this invention.

Alternatively, the derived molecules can have more than the 25 basepairs shown for each molecule, so long as the initial 25 contiguous basepairs remain. That is, once given the specific sequences disclosedherein, a person skilled in the art can synthesize molecules that, ineffect, “add” one or more base pairs to either or both ends in anyorder, creating molecules that are 26 or more base pairs in length andcontaining the original 25 contiguous base pairs.

The siRNA molecule may further comprise an immune stimulatory motif.Such motifs can include specific RNA sequences such as 5′-UGUGU-3′(Judge et al., Nature Biotechnology 23, 457-462 (1 Apr. 2005)),5′-GUCCUUCAA-3′ (Hornung et al., Nat. Med. 11,263-270(2005). See Kim etal., Mol Cell 24; 247-254 (2007). These articles are incorporated hereinby reference in their entireties. These are siRNA sequences thatspecifically activate immune responses through Toll-like receptor (TLR)activation or through activation of key genes such as RIG-I or PKR. Inone embodiment, the motif induces a TH1 pathway immune response. Inanother embodiment, the motif comprises 5′-UGUGU-3′, 5′-GUCCUUCAA-3′,5′-GGGxGG-3′ (where x is A, T, G and C), or CpG motifs 5′-GTCGTT-3′.

Pharmaceutical Compositions

The invention includes a pharmaceutical composition comprising an siRNAmolecule that targets a conserved region of the genome of a MERS-CoV anda pharmaceutically acceptable carrier. In one embodiment, the carriercondenses the molecules to form a nanoparticle. Alternatively, thecomposition may be formulated into nanoparticles. The compositions maybe lyophilized into a dry powder. In one particular embodiment, thepharmaceutically acceptable carrier comprises a polymeric nanoparticleor a liposomal nanoparticle.

In one embodiment, the composition comprises at least two differentsiRNA molecules that target one or more conserved regions of the genomeof a MERS-CoV and a pharmaceutically acceptable carrier. In one aspectof this embodiment, the gene sequences in the conserved regions of theMERS-CoV are critical for the viral infection of a mammal. In one aspectof this embodiment, mammal is a human, mouse, ferret, or monkey. Thecomposition can include one or more additional siRNA molecules thattarget still other conserved regions of the MERS-CoV genome. In oneaspect of this embodiment, a pharmaceutically acceptable carriercomprises a polymeric nanoparticle or a liposomal nanoparticle.

MRR1: CCCAGUGUUAUUGGUGUUUAUCAUA, (SEQ ID NO: 7)MRR2: GGGAUUUCAUGCUUAAAACAUUGUA, (SEQ ID NO: 8)MRR3: GGGUGCUAAUGGCAACAAGAUUGUU, (SEQ ID NO: 9)MRR4: CCCCAAAUUUGUUGAUAAAUACUAU, (SEQ ID NO: 10) andMRR5: CGGUUGCUUUGUAGAUGAUAUCGUU. (SEQ ID NO: 11)

In still another aspect of this embodiment, the siRNA molecules targetSpike viral gene expression. Such siRNA molecules include the following:

MSP1: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 12)MSP2: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 12)MSP3: CCGAAGAUGAGAUUUUAGAGUGGUU, (SEQ ID NO: 13)MSP4: CCCAGUUUAAUUAUAAACAGUCCUU, (SEQ ID NO: 14)MSP5: GGCUUCACUACAACUAAUGAAGCUU, (SEQ ID NO: 15)MSP6: CCCCUGUUAAUGGCUACUUUAUUAA, (SEQ ID NO: 16)MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, (SEQ ID NO: 17) andMSP8: GCCGCAUAAGGUUCAUGUUCACUAA. (SEQ ID NO: 18)

In a further aspect of this embodiment, the siRNA molecules are two ormore of the following:

MPL1: CGCAAUACGUAAAGCUAAAGAUUAU, (SEQ ID NO: 1)MPL2: GGGUGUUGAUUAUACUAAGAAGUUU, (SEQ ID NO: 2)MPL3: CGCAUAAUGGUGGUUACAAUUCUU, (SEQ ID NO: 3)MPL4: GGCUUCAUUUUAUUUCAAAGAAUUU, (SEQ ID NO: 4)MPL5: GCGCUUUUACAAAUCUAGAUAAGUU, (SEQ ID NO: 5)MPL6: CGCAUUGCAUGCCGUAAGUGUAAUU, (SEQ ID NO: 6)MRR1: CCCAGUGUUAUUGGUGUUUAUCAUA, (SEQ ID NO: 7)MRR2: GGGAUUUCAUGCUUAAAACAUUGUA, (SEQ ID NO: 8)MRR3: GGGUGCUAAUGGCAACAAGAUUGUU, (SEQ ID NO: 9)MRR4: CCCCAAAUUUGUUGAUAAAUACUAU, (SEQ ID NO: 10)MRR5: CGGUUGCUUUGUAGAUGAUAUCGUU, (SEQ ID NO: 11)MSP1: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 12)MSP2: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 12)MSP3: CCGAAGAUGAGAUUUUAGAGUGGUU, (SEQ ID NO: 13)MSP4: CCCAGUUUAAUUAUAAACAGUCCUU, (SEQ ID NO: 14)MSP5: GGCUUCACUACAACUAAUGAAGCUU, (SEQ ID NO: 15)MSP6: CCCCUGUUAAUGGCUACUUUAUUAA, (SEQ ID NO: 16)MSP7: CCCUGUUAAUGGCUACUUUAUUAAA, (SEQ ID NO: 17) andMSP8: GCCGCAUAAGGUUCAUGUUCACUAA. (SEQ ID NO: 18)

In another embodiment, the composition comprises an siRNA cocktail,MST^(PR1), wherein a first siRNA molecule comprises MPL1:CGCAAUACGUAAAGCUAAAGAUUAU (SEQ ID NO: 1) and a second siRNA moleculecomprises MRR1: CCCAGUGUUAUUGGUGUUUAUCAUA (SEQ ID NO: 7).

In another embodiment, the composition comprises an siRNA cocktail,MST^(PR2), wherein a first siRNA molecule comprises MPL2:GGGGUUGAUUAUACUAAGAAGUUU (SEQ ID NO: 19) and a second siRNA moleculecomprises MRR2: GGGAUUUCAUGCUUAAAACAUUGUA (SEQ ID NO: 8).

In another embodiment, the composition comprises an siRNA cocktail,MST^(RS2), wherein a first siRNA molecule comprises MRR2:GGGAUUUCAUGCUUAAAACAUUGUA (SEQ ID NO: 20) and a second siRNA moleculecomprises MSP2: GGCCGUACAUAUUCUAACAUAACUA (SEQ ID NO: 12).

In another embodiment, the composition comprises an siRNA cocktail,MST^(RS1), wherein a first siRNA molecule comprises MRR1:CCCAGUGUUAUUGGUGUUUAUCAUA (SEQ ID NO: 21) and a second siRNA moleculecomprises MSP1: GGCCGUACAUAUUCUAACAUAACUA (SEQ ID NO: 12).

In another embodiment, the composition comprises at least threedifferent siRNA molecules that target one or more conserved regions ofthe genome of a MERS-CoV and a pharmaceutically acceptable carrier. Inone aspect of this embodiment, the pharmaceutically acceptable carriercomprises a polymeric nanoparticle or a liposomal nanoparticle.

In another embodiment, the composition comprises an siRNA cocktail,MSTP^(RS1), wherein a first siRNA molecule comprises MPL1:CGCAAUACGUAAAGCUAAAGAUAU (SEQ ID NO: 22), a second siRNA moleculecomprises MRR1: CCCAGUGUUAUUGGUGUUUAUCAUA (SEQ ID NO: 7), and a thirdsiRNA molecule comprises MSP1: GGCCGUACAUAUUCUAACAUAACUA (SEQ ID NO:12).

In another embodiment, the composition comprises an siRNA cocktail,MST^(PRS2), wherein a first siRNA molecule comprises MPL2:GGGUGUUGAUUAUACUAAGAAGUUU (SEQ ID NO: 2) a second siRNA moleculecomprises MRR2: GGGAUUUCAUGCUUAAAACAUUGUA (SEQ ID NO: 8), and a thirdsiRNA molecule comprises MSP2: GGCCGUACAUAUUCUAACAUAACUA (SEQ ID NO:12).

In one aspect of all of these embodiments, the siRNA molecules comprise25 mer blunt-end siRNA molecules and the carrier comprises aHistidine-Lysine copolymer or Spermine-Lipid Conjugate and cholesterol.

Pharmaceutically Acceptable Carriers

Pharmaceutically acceptable carriers include saline, sugars,polypeptides, polymers, lipids, creams, gels, micelle materials, andmetal nanoparticles. In one embodiment, the carrier comprises at leastone of the following: a glucose solution, a polycationic binding agent,a cationic lipid, a cationic micelle, a cationic polypeptide, ahydrophilic polymer grafted polymer, a non-natural cationic polymer, acationic polyacetal, a hydrophilic polymer grafted polyacetal, a ligandfunctionalized cationic polymer, a ligand functionalized-hydrophilicpolymer grafted polymer, and a ligand functionalized liposome. Inanother embodiment, the polymers comprise a biodegradablehistidine-lysine polymer, a biodegradable polyester, such as poly(lacticacid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolicacid) (PLGA), a polyamidoamine (PAMAM) dendrimer, a cationic lipid, or aPEGylated PEI. Cationic lipids include DOTAP, DOPE, DC-Chol/DOPE, DOTMA,and DOTMA/DOPE. In still another embodiment, the carrier is ahistidine-lysine copolymer that forms a nanoparticle with the siRNAmolecule, wherein the diameter of the nanoparticle is about 100 nm toabout 400 nm.

In one embodiment, the carrier is a polymer. In one aspect of thisembodiment, the polymer comprises a histidine-lysine copolymer (HKP).Such copolymers are described in U.S. Pat. Nos. 7,070,807 B2, 7,163,695B2, and 7,772,201 B2, which are incorporated herein by reference intheir entireties. In one aspect of this embodiment, the HKP comprisesthe structure (R)K(R)-K(R)-(R)K(X), where R=KHHHKHHHKHHHKHHHK (SEQ IDNO: 23), K=lysine, and H=histidine.

In another embodiment, the carrier is a liposome. In one aspect of thisembodiment, the liposome comprises a cationic lipid conjugated withcholesterol. In a further aspect, the cationic lipid comprises aspermine head and one or two oleyl alcoholic tails. Examples of suchmolecules are disclosed in FIG. 8. In a further aspect, the liposomecomprises Spermine-Liposome-Cholesterol conjugate (SLiC).

Methods of Use

The invention also includes methods of using the siRNA molecules andpharmaceutical compositions containing them to prevent or treat MERS-CoVdisease. A therapeutically effective amount of the composition of theinvention is administered to a subject. In one embodiment, the subjectis a mammal such as a mouse, ferret, monkey, or human. In one aspect ofthis embodiment, the mammal is a laboratory animal, such as a rodent. Inanother aspect of this embodiment, the mammal is a non-human primate,such as a monkey. In still another aspect of this embodiment, the mammalis a human. As used herein, a “therapeutically effective amount” is anamount that prevents, reduces the severity of, or cures MERS disease.Such amounts are determinable by persons skilled in the art, given theteachings contained herein. In one embodiment, a therapeuticallyeffective amount of the pharmaceutical composition administered to ahuman comprises about 1 mg of the siRNA molecules per kilogram of bodyweight of the human to about 5 mg of the siRNA molecules per kilogram ofbody weight of the human. Routes of administration are also determinableby persons skilled in the art, given the teachings contained herein.Such routes include intranasal administration and airway instillation,such as through use of an airway nebulizer. Such routes also includeintraperitoneal, intravenous, and subcutaneous administration.

EXAMPLES

We selected Papain-like protease (PL^(PRO)), RNA-dependent RNApolymerase (RdRp), Spike(S) protein and some of other structure genes(such as M and N protein) and non-structure genes (such as nsp-2, nsp-10and nsp-15) of MERS-CoV as the targets for an siRNA cocktail-mediatedtherapeutic approach. The present invention provides siRNA moleculesthat target a conserved region of MERS-CoV, wherein the siRNA moleculesinhibit expression of those genes of MERS-CoV. In a preferredembodiment, the molecule comprises a double-stranded sequence of 17, 18,19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In one aspect ofthis embodiment, the siRNA molecule has blunt ends, or has 3′ overhangsof one or more nucleotides on both sides of the double-stranded region.The siRNA cocktail of the invention contains two, three, four, or moresequences targeting those genes of MERS-CoV.

Example 1 MERS-CoV Viral Structure and Protein Function

MERS-CoV is enveloped single-stranded positive sense RNA viruses withgenomes of 30,119 nt. The genome structure of MERS-CoV is similar toother coronaviruses, with the 5′ two-thirds of the genome encoding thenon-structural proteins (NSPs) required for viral genome replication,the remaining 3′ third of the genome encoding the structural genes thatmake up the virion (spike, envelope, membrane, and nucleocapsidproteins), and four accessory genes interspersed within the structuralgene region (FIG. 1A). At the 5′ end of the genome there is a leadersequence (67nt), which is followed by an untranslated region (UTR). Atthe 3′ end of the RNA genome there is another UTR, followed by a poly(A)sequence of variable length. Transcription-regulatory sequences (TRS 5′AACGAA 3′) are found at the 3′ end of the leader sequence and atdifferent positions upstream of genes in the genomic 3′-proximal domainof MERS-CoV. The MERS-CoV genome contains at least 10 predicted openreading frames (ORFs): ORF1a, ORF1b, S, 3, 4a, 4b, 5, E, M and N withsixteen predicted nonstructural proteins being encoded by ORF1a/b.Several unique group-specific ORFs that are not essential for virusreplication are encoded by MERS-CoV. The functions of thesegroup-specific ORFs are unknown; however, by analogy to othercoronaviruses, they may encode structural proteins or interferonantagonist genes. Open reading frames ORF2, -6, -7 and-8a are translatedfrom subgenomic mRNAs predicted to encode the four canonical structuralgenes: a 180/90-kDa spike glycoprotein (S), a˜23-kDa membraneglycoprotein(M), a small envelope protein (E) and a˜50-kDanucleocapsidprotein (N), respectively (FIG. 1B-C).

Example 2 MERS-CoV Viral Genes and RNAs

Similar to other RNA viruses, coronavirus replicate in the hostcytoplasm. The replication process is initiated by the viral particleafter binding with specific cellular receptors, known as S-proteinmediated binding. The receptor for MERS-CoV was recently identified asdipeptidyl peptidase 4 (DDP4, also known as CD26), a protein withdiverse functions in glucose homeostasis, T-cell activation,neurotransmitter function, and modulation of cardiac signaling. DPP4 isexpressed in a variety of cell types, including endothelial cells(kidney, lung, small intestine, spleen) hepatocytes, enterocytes,activated leukocytes, testes, prostate and cells of the renal glomeruliand proximal tubules. DPP4 recognition is mediated by thereceptor-binding domain (RBD, amino acids E367-Y606). Following virusentry, the coronavirus genome, a positive sense, capped andpolyadenylated RNA strand, is directly translated, resulting in thesynthesis of coronavirus replicase gene-encoded NSPs. Coronavirus NSPsare translated as two large polyproteins harboring proteolytic enzymes,namely papain-like and chymotrypsin-like proteinases that extensivelyprocess coronavirus polyproteins to liberate up to 16 NSPs (nsp 1-16).After entering into the cell the virus specially modulates the innateimmune response, antigen presentation, mitogen-activated protein kinase(FIG. 2).

Example 3 Design siRNA Targeting Key Genes of MERS-CoV

Using our specific algorithm, we have designed multiple siRNA sequences,including both 25-mer and 23-mer oligos. Table I. siRNA sequences,25-mer blunt-end oligos and 23-mer sticky-end oligos, targeting variousviral RNA Table II. siRNA sequences, 25-mer blunt-end oligos and 23-mersticky-end oligos, targeting various viral RNA Table III. We selectedthe most potent siRNA oligos, 25-mer blunt-end oligos and 23-mersticky-end oligos, targeting various viral proteins and genes. Asdemonstrated in the FIG. 3, we are specifically targeting critical viralgenes: Papain like protein (PL^(pro)) specific siRNA, total 6 activesiRNAs (MPL1-6); RNA dependent RNA protease (RDRP) specific siRNA, total5 active siRNAs (MRR1-5) and Spike protein specific siRNA, total 8active siRNAs (MSP1-8).

Example 4 Cell Culture Based Screening for Potent Anti-MERS CoV siRNAOligos

Firstly, to identify the most potent siRNA for silencing MERS-CoV genesin Vero cell culture experiments, we used psiCheck plasmid carryingMERS-CoV gene sequences.

Secondly, we used Vero cell infected with real MERS-CoV to test theselected siRNA for their anti-MERS CoV infecting activity.

-   A. Subcloning MERS-CoV virus gene fragments as surrogates for siRNA    potency examination in Vero cells In order to investigate the    degrading effect of siRNA candidates on targeted MERS-CoV genes, we    used a dual luciferase reporter vector, psiCHECK-2, with gene    fragments of Papain like viral protein (nsp5), Conoravirus    endopeptidase C30 (nsp6), RNA synthesis protein (nsp10),    RNA-dependent RNA polymerase (nsp12), and structure proteins S, E, M    and N. psiCHECK-2 Vectors are designed to provide a quantitative and    rapid approach for initial optimization of RNA interference (RNAi).    The vectors enable monitoring of changes in expression of a target    gene fused to a reporter gene. The DNA fragments of nsp5, nsp6,    nsp10, nsp12 and structure proteins S, E, M and N were amplified by    PCR with specific primers to those genes, and then cloned into the    multiple cloning sites of psiCHECK-2 Vector. In this vector, Renilla    Luciferase is used as a primary reporter gene, and the siRNA    targeting genes located downstream of the Renilla translational stop    codon.

Vero cells were seeded in 96-well plates and incubated for 12 h. Thereporter plasmids (recombinant vectors) psi-nsp5, psi-nsp6, psi-nsp10,psi-nsp12, psi-S, psi-E, psi-M and psi-N, and siRNA candidates wereco-transfected into Vero cells using Lipofectamine 2000 in the DMEMwithout FBS. The blank psi vector is taken as a negative control. Sixhours post-transfection, the media was replaced with the DMEMsupplemented with 10% FBS. 18, 24, 36 and 48 h post-transfection theactivity of the firefly luminescence and Renilla Luciferase in each wellwas detected using the Dual Luciferase Kit. The siRNA candidatesdramatically decreased luciferase activity which indicates that siRNAcould greatly inhibit the expression of the target genes of MERS-CoVwere selected for the assay of infection with MERS-CoV in vitro.

-   B. Infection of Vero cells with MERS-CoV To investigate whether the    real MERS-CoV mRNAs for nsp5, nsp6, nsp10, nsp12 and structure    proteins S, E, M and N can be directly degraded by the specific    mechanism of RNA interference (RNAi), Vero cells were seeded in    24-well plate and transfected with selected therapeutic single siRNA    or siRNA combination candidates using Lipofectamine 2000 in the DMEM    without FBS when cell monolayer reached 80% confluency. The    transfection efficacy control is Cy3 labeled siRNA. PBS was taken as    a negative control. An siRNA with the sequence unrelated to MERS-CoV    was used as another negative control. 24 h post-transfection the    media containing the transfection reagent was replaced with fresh    media supplemented with 2% FBS, and cells were infected with    MERS-CoV. One hour post-infection, the inoculation solution was    replaced with DMEM supplemented with 10% FBS. 24 h post-infection,    cells were harvested for RNA isolation and 5′-rapid amplification of    cDNA ends (5′-RACE). In the other parallel experiment, at 24, 48 and    72 h post-infection, the cell supernatants were harvested for viral    titer determination. All experiments were performed under Biosafety    level-2 condition.

The viral RNA were extracted from the cell supernatants, and theone-step quantitative real-time PCR were performed with forward, reverseprimers and TaqMan probe specific to the MERS-CoV isolate FRA/UAE spikeprotein. The total RNA from the harvested cells was extracted, and5′-RACE assays were carried out with gene-specific primers for cDNAproducts of nsp5, nsp6, nsp10, nsp12 and structure proteins S, E, M andN. The single siRNAs or siRNA combinations with high protectionefficiency were selected for in vivo studies.

Example 5 HKP/siRNA nanoparticle and pulmonary delivery

Histidine-Lysine co-polymer (HKP) siRNA nanoparticle formulations can beestablished by mixing together aqueous solutions of HKP and siRNA in 4:1ratio by a molecular weight (N/P). A typical HKP/siRNA formulation willprovide nanoparticles in average size in 150 nm in diameter (FIG. 4A).The self-assembled HKP/siRNA nanoparticles can be resuspended in aqueoussolution, lyophilized into dry powder, and then resuspended in RNasefree water (FIG. 4B). After oral-trachial administration of HKP/siRNA () nanoparticles to the mouse respiratory track we were able to observefluorescent siRNA in the upper (bronchi), and lower airway (alveoli)(FIG. 4C). We compared the efficacy of RNAi of cyclophiline B in thelung after oraltrachial deliveries of three different doses of siRNAwith HKP, DOTAP and D5W. HKP-mediated delivery demonstrated theefficient RNAi of the target gene at the 20 μg dose (FIG. 5).

Example 6 HKP/siRNA Formulation for Intraperitoneal Delivery

During evaluation of prophylaxis and therapeutic benefit of siRNAinhibitors against influenza infection, we tested HKP/siRNA formulationthrough intraperitoneal administration, using different dosage andregimens. Based on the observations of these treatment results, we foundthat the prophylactic effect of HKP/siRNA (two siRNAs arespecific toinfluenza genes) exceed the effect of Ribovirin (FIG. 6). Similarly, thetherapeutic effect of HKP/siRNA (two siRNAs are specific to influenzagenes) is greater than Tamiflu® effect (FIG. 7). Due to the fact thatboth influenza and MERS infections occur in the human respiratorysystem, we are envisioning that the similar therapeutic approach, suchas the HKP/siRNA therapeutics, can be applied for treatment of MERSsince we observed the positive therapeutic benefit.

Example 7 SLiC/siRNA Nanoparticle

SLiC Liposome Preparation. Regular methods were tried at first toprepare liposomes with newly synthesized SLiC molecules, such as thinfilm method, solvent injection and so on without much success. NorbertMaurer et al reported a method of liposome preparation in which siRNA oroligonucleotide solution was slowly added under vortexing to the 50%ethanol solution (v/v) of liposome and ethanol was later removed bydialysis. The nanoparticles thus derived were small in size andhomogeneous. In this method, siRNA was directly wrapped by cationiclipids during formation of liposome, while in most other methods siRNAor nucleic acid molecules are loaded (or entrapped) into preformedliposome, such as Lipofectamine 2000.

Lipids dissolved in ethanol are in so-called metastable state in whichliposomes are not very stable and tend to aggregate. We then preparedun-loaded or pre-formed liposomes using modified Norbert Maurer'smethod. We found that stable liposome solution could be made by simplydiluting ethanol to the final concentration of 12.5% (v/v). Liposomeswere prepared by addition of lipids (cationic SLiC/cholesterol, 50:50,mol %) dissolved in ethanol to sterile dd-H₂O. The ethanolic lipidsolution needs to be added slowly under rapid mixing.

Slow addition of ethanol and rapid mixing were critical for the successin making SLiC liposomes, as the process allows formation of small andmore homogeneous liposomes. Unlike conventional methods, in which siRNAsare loaded during the process of liposome formulation and ethanol orother solvent is removed at end of manufacturing, our SLiC liposomeswere formulated with remaining ethanol still in the solution so thatliposomes were thought to be still in metastable state. When siRNAsolution was mixed/loaded with liposome solution cationic groups, lipidswill interact with anionic siRNA and condense to form core. SLiCliposomes' metastable state helped or facilitated liposome structuretransformation to entrap siRNA or nucleic acids more effectively.Because of the entrapment of siRNA, SLiC liposomes become more compactand homogeneous.

Physiochemical Characterization of SLiC Liposome. After the liposomeformation, we have developed an array of assays to characterize thephysicochemical properties of SLiC liposome, including particle size,surface potential, morphology study, siRNA loading efficiency andbiological activity, etc. The particle size and zeta-potentials of SLiCliposomes were measured with Nano ZS Zeta Sizer (Malvern Instruments,UK). Each new SLiC liposome was tested for particle size andzeta-potential when ethanol contents changed from 50% to 25% and to12.5%. Data were derived from formulations of different ethanolcontents. All SLiC liposomes were prepared at 1 mg/ml in concentrationand loaded with siRNA (2:1, w/w). Each of SLiC Liposomes was composed ofcationic SLiC and cholesterol dissolved in ethanol at 12.5%, e.g. TM2(12.5). The average particle sizes of three sequential measurements andthe average zeta-potentials of three sequential measurements wereillustrated in Table IV.

Further analysis of the physiochemical perimeters of the SLiC liposomesuggested that ethanol concentrations were positively proportional toparticle sizes (the lower of ethanol concentration, the smaller ofparticle sizes), but negatively proportional to zeta-potential (thelower of ethanol concentration, the higher of zeta-potential at the sametime). The higher surface potential will render particles more stable insolution. In addition to stability in solution, data shown later alsoindicated that toxicity was lower with lower ethanol concentration, too.Therefore, to put all factors together, ethanol concentration of 12.5%(v/v) was selected as solvent to suspend cholesterol as well as SLiCinto the master working stock solution before they were used to makeliposome formulations.

In contrast to bare SLiC liposome formulation, liposomes particle sizesbecame much smaller when they were loaded with siRNA at 2:1 (w/w)resulting in particle sizes in the range of 110 to 190 nm in diameterand much lower PDI values. Conventional consideration of liposomalstructure dictates that siRNA is loaded or interacted with cationiclipids through electrostatic forces and liposomes wraps siRNA to formspherical particles in shape in order to reduce surface tension. As theresult, the liposomes particle sizes became much smaller after loadedwith siRNA. Liposomes formulated with siRNA also have lower surfacecharge, which could be explained by neutralizing effect from loadedsiRNA.

Example 8 Airway Delivery with Mouse Model

Human host-cell dipeptidyl peptidase 4 (hDPP4) has been shown to be thereceptor of MERS-CoV. However, mouse is not a suitable small-animalmodel for MERS-CoV as it has no receptor being recognized and bound bythe virus. In this study, the mice were sensitized to MERS-CoV infectionby transduction with Adenoviral or Lentiviral vector expressing hDPP4 inthe respiratory tract. This mouse model was used to investigate theefficiency of the siRNA on inhibiting the MERS-CoV infection in vivo.The siRNA combination candidate was delivered by encapsidated withHKP-SLiC nanoparticle system. We performed all mouse studies underBiosafety level-3 conditions.

All BALB/c mice were 18 weeks old and tested as specific pathogen-freeat the beginning of this study. To develop the susceptibility toMERS-CoV, 30 mice of Adenoviral vector group and 30 mice of Lentiviralvector group were transduced with Adenoviral and Lentiviral vectorexpressing hDPP4, respectively. Another 20 mice were transduced withempty Adenoviral or Lentiviral vector as the control. For the Adenoviralvector group, hDDP4 gene was cloned into the Ad5. Then MLE 15 cells weretransduced with Ad5-hDDP4 at an MOI of 20. The supernatant werecollected at 48 h post-infection. The mice were transduced intranasallywith 10⁸ pfu of Ad5-hDDP4. For the Lentiviral vector group, hDDP4 genewas cloned into the plasmid pWPXLd. Then, pWPXLd-hDPP4, along withpackaging vector, psPAX2, and envelope vector, pMD2.G, wasco-transfected into packaging cell line HEK 293T using calcium phosphatemethod. At 48 h post-transfection, the constructed viral vector washarvested and purified, and transducted with CHO cells. The lentiviruswas harvested and concentrated. The mice were transduced intranasallywith lentivirus expressing hDPP4 at titers of 10⁸ transducing units/ml(TU/ml).

After confirming the hDPP4 was expressed in the respiratory tract of themice by western blot, the Adenoviral and Lentiviral vector groups werefurther divided into prophylactic, therapeutic and control subgroup withten mice in each subgroup. Ten mice from Ad5-hDDP4 or psPAX2-hDDP4prophylactic subgroup were intranasally inoculated with siRNAcombination encapsidated with HKP-SLiC nanoparticle system 24 h beforeinoculation. 24 h later, all eighty mice including transduced with emptyvector were infected intravenously with 10⁵ pfu of MERS-CoV. Theprophylactic, therapeutic and control subgroup were intranasallyinoculated with siRNA or PBS at 0, 24, 48, 72 and 96 h post-infection.

All mice were weighed and the survivors of each subgroup were counteddaily. The nasal washes were collected at 1, 3, 5, 7, 9, and 14 daypost-infection for the viral titration. Two infected mice from eachgroup were sacrificed at 3 and 5 day post-infection, respectively. Thetissue collection, including lung, trachea, spleen, liver, heart, brainand kidney, were collected for pathological and virological study.

To determine the viral titers, the tissue samples were homogenized inDMEM, and clarified by centrifugation. Both tissue suspensions and nasalwashes were10-fold serially diluted. The dilutions were added to theVero cells monolayers grown in 96-well plates. The cytopathic effects(CPEs) were observed on day 3 post-infection, and the TCID₅₀ wascalculated by the Reed-Muench method.

To investigate the efficiency of siRNA candidates in inhibiting viralgene expression, the total RNAs were extracted from the tissues and theone-step quantitative real-time PCR were performed with forward, reverseprimers and TaqMan probe specific to the conserved region of nsp12(RNA-dependent RNA polymerase) of MERS-CoV.

Example 9 Intraperitoneal siRNA Nanoparticle Solution

In vivo administration of siRNAs. The in vivo experiments were conductedusing 6-8 week old female mice. For inoculation, allantoic fluidcontaining the virus at a dose of 5×10⁴ EID₅₀/mL was used. Theinfectious activity of the virus in allantoic fluid was determined invivo by titration of lethality. Titers of the virus were calculatedusing the Reed and Muench method. Non-infected mice that were kept inthe same conditions as the infected animals were used as a negativecontrol. Virus was administered to the animals intranasally under alight ether anesthesia. Each group of animals contained 15 mice. siRNA(1:1 ratio of siRNAs 89 and 103) complexed with PAA as described above,was administered to the animals at the dose of 1-10 mg/kg of bodyweight. siRNA was administered intraperitoneally (200 ul per injection).Control animals received PAA without siRNAs.

Animals were observed for 14 days post inoculation. The mortality of theanimals in control and experimental groups was registered daily. Themortality percentage (M) was calculated in each group as: M=N/Nt where:N—the number of animals died within14 days after infection; Nt—the totalnumber of animals in the group. The index of protection (IP) wascalculated as: IP=((Mc−Me)/Mc)×100%, where: Mc and Me—percentage ofmortality in control and experimental groups, correspondingly. Themedian day of death (MDD) within 14 days was calculated as: MDD=(Σ ND)/Nt, where: N—the number of animals surviving D days; Nt—total numberof animals in the group Tamiflu® (oseltamivir phosphate, Roche,Switzerland) was used as a reference compound. It was administered at adose of 25 mg/kg by the same protocol.

The intraperitoneal administration could be a viable alternative,especially in patients with severe influenza with low gas-exchangevolume and/or those on mechanical lung ventilation. Since siRNAs of thesame length show similar properties (charge, hydrophobicity, molecularweight etc) and since siRNAs can be rapidly designed and manufactured,it is feasible that nanoparticle-mediated siRNA delivery may form anintermediate therapeutic strategy in treating rapidly emerging influenzavirus strains with high mortality rates that do not respond to existingtherapies, while vaccines to protect the general population are underdevelopment. The siRNA cocktail demonstrated herein may providesignificant value as a prophylactic/therapeutic with broadanti-influenza strain coverage and this coverage may well extend to asyet unidentified Influenza strains that may emerge in the future. Asstated in the Example 6, the therapeutic benefit we observed during thestudy using siRNA approach against influenza viral infection hasprovided a good example to follow: the HKP/siRNA nanoparticle deliverythrough IP route or respiratory route, targeting the conservativeregions of the critical viral gene sequences, and siRNA cocktail design,etc.

We demonstrated in this study that polymeric nanoparticle-mediateddelivery of a combination of two siRNAs, via IP administration, canresult in a potent antiviral effect in the viral-challenged animals.Histidine Lysine Co-Polymer (HKP) nanoparticle-mediated siRNA deliveryhas been well validated through multiple routes with various animalmodels (17). We recently completed a full scale safety study forHKP-siRNA nanoparticle formulation via subcutaneous administration intoboth mouse and monkey models (data not shown). When HKP-siRNA103/105formulation was IP administrated (10 mg/kg/day), a prophylactic andtherapeutic benefit greater than that observed with Ribavirin (75mg/kg/day) in protecting mice from exposure to a 2× LD50 dose of thevirus. (Ribavirin is manufactured by multiple companies in the UnitedStates: Copegus produced by Genentech (member of the Roche group),Rebetol by Merck Sharp & Dome, a subsidary of Merck & Co., Inc., andRibasphere by Kadmon Pharmaceuticals (orginaily by Three RiversPharmaceuticals which was acquired by Kadmon Pharmaceuticals). Inaddition, several companies, including Sandoz and Teva pharmaceuticals,produce generic ribavirin.) The data obtained suggests that IP injectionof peptide nanoparticles containing siRNAs or of a polycationic deliveryvehicle carrying siRNAs can both ameliorate the lethality induced byInfluenza infection in mice and therefore may suggests the ability toovercome some of these barriers. The amphiphilic poly(allylamine) (PAA)formed polymeric micelles (PM) has been evaluated for siRNA delivery viathe GI tract, resulting in efficient siRNA delivery andendosome/lysosome release. PAA and siRNA can be self-assembled intocomplexes with nano-sized diameters (150-300 nm) and cationic surfacecharge (+20 to 30 mV). When we IP administered PAA-siRNA89/103formulation (10 mg/kg) a therapeutic antiviral activity was observedequivalent to that of Tamiflu (25 mg/kg). These results clearlydemonstrated that polymeric nanoparticle delivery of siRNA combinationsmay provide a prophylactic/therapeutic response against newly emergentstrains of influenza virus. A similar approach can be considered for aMERS siRNA therapy.

Example 10 Effects on Innate Immunity in Lung

To evaluate the cytokine response after HKP/siRNA formulationadministration to the mouse airway, we collected the lung lavage samplesfrom the Balb/c mice treated with intratracheal instillation ofHKP/siRNA aqueous solution (the cohort and dosage described in Table A).The lavage samples were further measured for the TNF-α, IL-6 and IFN-αchanges before and after the treatment using commercial available ELISAassay (FIG. 10). We first established a standard curve (using Lowrymethod) of the protein concentration and then measured the proteinconcentrations of each collected sample, where the STP705 stands forHKP/siRNA groups with different siRNA concentrations (FIG. 11). Based onthe standard curves for TNF-α, IL-6 and IFN-α we established using thecommercial kits (FIG. 12A, FIG. 13A and FIG. 14A), we measured theTNF-α, IL-6 and IFN-α cytokine levels of each collected samples (FIG.12B, FIG. 13B and FIG. 14B). With comparisons between the normal mouselungs and LPS treated mouse lungs, and HKP/siRNA treated lungs, we foundthat (1) HKP/siRNA treatment has little impact on the lung TNF-α levelchanges (FIG. 12B); (2) Various HKP/siRNA formulations with differentsiRNA concentrations can induce IL-6 level elevation (FIG. 13B); (3)There is no significant changes of the IFN-α levels with the HKP/siRNAformulation treatments.

TABLE A Effect of siRNA-HKP nanoparticles on innate immunity at lungExperiment design Group Animal# composition 1, Normal 5 — 2, Blank 50.9% NS 3, HKP 5 160 μg HKP 4, siRNA 5 40 μg siRNA{circle around(1)}+{circle around (2)} 5, HKP/siRNA high 5 160 μg HKP + 40 μg siRNA 6,HKP/siRNA low 5  80 μg HKP + 20 μg siRNA 7, HKP/siRNA 5 20 μg STP705 8,LPS 5 5 mg/kg

Example 11 Non-Human Primate Study

Currently, there is neither an effective vaccine nor drug available forprophylatic or therapeutic strategy. Recently, rhesus macaque has beendeveloped as a model for MERS-CoV using intratracheal inoculation.Similar to human, the infected monkeys showed clinical signs of disease,virus replication, and histological lesions, indicating that rhesusmacaque is a good model for evaluation of vaccine and antiviralstrategies against MERS-CoV infection.

To investigate the efficiency of the siRNA on protecting and healingfrom MERS-CoV infection, we plan to perform the non-human primate studyin rhesus macaques. The siRNA cocktail candidate will be encapsidatedwith HKP-SLiC nanoparticle system, and administered intratracheally.This monkey study should be carried out under Biosafety Level-3condition.

All rhesus monkeys should be 2-3 years old at the beginning of thisstudy. At the beginning, all monkeys need to be tested negative forMERS-CoV. Twelve monkeys should be divided into threegroups—prophylactic, protection, and control group with four animals ineach group. Four monkeys of prophylactic group should be intratracheallyinoculated with siRNA combination encapsidated in HKP-SLiC nanoparticlesystem using a nebulizer. 24 h later, all twelve monkeys should beintratracheally inoculated with 6.5×10⁷ TCID₅₀ of MERS-CoV in 1 mL. Theprophylactic and protection groups should be continuously inoculatedwith siRNA combination at 0, 24, 48, 72 and 96 h post-infection usingthe nebulizer. The control group will be inoculated with PBS at the sametime points.

All monkeys will be observed twice daily for the symptoms and mortality.Chest X-rays need to be performed 1 day pre-infection and 3, and 5 daypost-infection. Oropharyngeal, nasal, and cloacal swabs should becollected at 1, 3, 5, 7, 9, 14, 21, and 28 day post-infection for theviral titration. Two infected monkeys from each group will be sacrificedon the day 3 post-infection. The tissue including lung, trachea, spleen,liver, heart, brain, kidney, and colon tissue will be collected forpathological and virological study.

The viral titers determination in the tissue and swab samples should beperformed as described in Example 2. To investigate the efficiency ofsiRNA candidates on inhibiting viral gene expression, the total RNA willbe extracted from the tissues and the one-step quantitative real-timePCR were performed.

To investigate the efficiency of siRNA candidates on inhibiting viralprotein expression, the total RNA will be extracted from the tissues andthe one-step quantitative real-time PCR will be performed as describedin Example 8.

REFERENCES

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The disclosures of all publications identified herein, including issuedpatents and published patent applications, and all database entriesidentified herein by url addresses or accession numbers are incorporatedherein by reference in their entirety.

Although this invention has been described in relation to certainembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

TABLE 1 Predicted 25 mer siRNA targeting MERS NC019843.325mer blunt ended sequences 23 mer Sequences passing all SEQ ID StartProtein metrics and BLAST search (allows SEQ ID SiRNA sequence NO: BaseCoded for 2 base overhang on 21mer) NO: GGCUCAUUGCUUGUGAAAAUCCAUU  24 1times at 555 NSP1 GCUUGUGAAAAUCCAUUCAUGGUUA  25 1 times at 563 NSP1CCAUUCAUGGUUAACCAAUUGGCUU  26 1 times at 575 NSP1CGAACUUGUCACAGGAAAGCAAAAU  27 1 times at 679 NSP1GCAAAAUAUUCUCCUGCGCAAGUAU  28 1 times at 697 NSP1CCCCAUUCCACUAUGAGCGAGACAA  29 1 times at 744 NSP1GGCAAAUAUGCCCAGAAUCUGCUUA  30 1 times at 815 NSP1GCAAAUAUGCCCAGAAUCUGCUUAA  31 1 times at 816 NSP1CCCAGAAUCUGCUUAAGAAGUUGAU  32 1 times at 825 NSP1CCCAGAAUCUGCUUAAGAAGUUG  952 CCAGAAUCUGCUUAAGAAGUUGAUU  33 1 times at826 NSP1 GCUUAAGAAGUUGAUUGGCGGUGAU  34 1 times at 835 NSP1CGGUGAUGUCACUCCAGUUGACCAA  35 1 times at 853 NSP1GGUGAUGUCACUCCAGUUGACCAAU  36 1 times at 854 NSP1GGAAAACCCAUUAGUGCCUACGCAU  37 1 times at 896 NSP2CCCAUUAGUGCCUACGCAUUUUUAA  38 1 times at 902 NSP2CCAUUAGUGCCUACGCAUUUUUAAU  39 1 times at 903 NSP2GGAUGGAAUAACCAAACUGGCUGAU  40 1 times at 934 NSP2CGUCGCAGCACGUGCUGAUGACGAA   41 1 times at 970 NSP2GCUGAUGACGAAGGCUUCAUCACAU  42 1 times at 983 NSP2CGUUCCAUAUCCUAAGCAAUCUAUU  43 1 times at 1054 NSP2CCAUAUCCUAAGCAAUCUAUUUUUA  44 1 times at 1058 NSP2CCUAAGCAAUCUAUUUUUACUAUUA  45 1 times at 1064 NSP2CCUCCUCACUAUUUUACUCUUGGAU  46 1 times at 1124 NSP2CGUUUCUGACUUGUCCCUCAAACAA  47 1 times at 1189 NSP2GGUAAGGAGUCACUUGAGAACCCAA  48 1 times at 1235 NSP2CCAACCUACAUUUACCACUCCGCAU  49 1 times at 1256 NSP2CCUACAUUUACCACUCCGCAUUCAU  50 1 times at 1260 NSP2GCUAUCCAAGGGUUUGCCUGUGGAU  51 1 times at 1328 NSP2GGGUUUGCCUGUGGAUGUGGGGCAU  52 1 times at 1337 NSP2GCCUGUGGAUGUGGGGCAUCAUAUA  53 1 times at 1343 NSP2GGAUGUGGGGCAUCAUAUACAGCUA  54 1 times at 1349 NSP2GGCGUAGCUUACGCCUACUUUGGAU  55 1 times at 1559 NSP2GCCUACUUUGGAUGUGAGGAAGGUA  56 1 times at 1571 NSP2CCUAGAGCUAAGUCUGUUGUCUCAA  57 1 times at 1610 NSP2CCUUAACUUUGUGGGAGAGUUCGUU  58 1 times at 1726 NSP2GGGAGAGUUCGUUGUCAACGAUGUU  59 1 times at 1738 NSP2GCCGGCCCAUUCAUGGAUAAUGCUA  60 1 times at 1880 NSP2CCGGCCCAUUCAUGGAUAAUGCUAU  61 1 times at 1881 NSP2CGGCCCAUUCAUGGAUAAUGCUAUU  62 1 times at 1882 NSP2GGCCCAUUCAUGGAUAAUGCUAUUA  63 1 times at 1883 NSP2GCCCAUUCAUGGAUAAUGCUAUUAA  64 1 times at 1884 NSP2GCCCAUUCAUGGAUAAUGCUAUU  953 CCCAUUCAUGGAUAAUGCUAUUAAU  65 1 times at1885 NSP2 CCCAUUCAUGGAUAAUGCUAUUA  954 GCUAUUAAUGUUGGUGGUACAGGAU  66 1times at 1901 NSP2 CGCCAUUACUGCACCUUAUGUAGUU  67 1 times at 1936 NSP2CGCCAUUACUGCACCUUAUGUAG  955 GCUCACAGCGUGUUGUACAGAGUUU  68 1 times at2048 NSP2 GCGUGUUGUACAGAGUUUUUCCUUA  69 1 times at 2055 NSP2CGUGUUGUACAGAGUUUUUCCUUAU  70 1 times at 2056 NSP2GUGUUGUACAGAGUUUUUCCUUA  956 GGCGACUUUAUGUCUACAAUUAUUA  71 1 times at2186 NSP2 GGCGACUUUAUGUCUACAAUUAU  957 CCAAACUGCUGUUAGUAAGCUUCUA  72 1times at 2218 NSP2 GCUGUUAGUAAGCUUCUAGAUACAU  73 1 times at 2225 NSP2CUGUUAGUAAGCUUCUAGAUACA  958 GCAACAUUUAACUUCUUGUUAGAUU  74 1 times at2267 NSP2 AACAUUUAACUUCUUGUUAGAUU  959 CCUAUGUGUACACUUCACAAGGGUU  75 1times at 2325 NSP2 GGAACCUAUUACUGUGUCACCACUA  76 1 times at 2504 NSP2GGUUGAAACUGUUGUGGGUCAACUU  77 1 times at 2653 NSP2GCAAACUAAUAUGCAUAGUCCUGAU  78 1 times at 2680 NSP2GGUGACUAUGUCAUUAUUAGUGAAA  79 1 times at 2714 NSP2GGGAGGUGCACCUGUAAAAAAAGUA  80 1 times at 2830 NSP2CGAGUACAACAUUCAUGCUGUAUUA  81 1 times at 2908 NSP3GCUGUAUUAGACACACUACUUGCUU  82 1 times at 2924 NSP3GGAGUUUGCUGACGUAGUAAAGGAA  83 1 times at 2995 NSP3GCGUGGAAUGCCGAUUCCAGAUUUU  84 1 times at 3049 NSP3GGAAUGCCGAUUCCAGAUUUUGAUU  85 1 times at 3053 NSP3CCAGAUUUUGAUUUAGACGAUUUUA  86 1 times at 3065 NSP3CGAUUUUAUUGACGCACCAUGCUAU  87 1 times at 3082 NSP3CCCGUCGAGUGUGACGAGGAGUGUU  88 1 times at 3164 NSP3CGAGUGUGACGAGGAGUGUUCUGAA  89 1 times at 3169 NSP3GGCUUCAGAUUUAGAAGAAGGUGAA  90 1 times at 3199 NSP3GCUUCAGAUUUAGAAGAAGGUGAAU  91 1 times at 3200 NSP3CGACGAGUGGGCUGCUGCAGUUGAU  92 1 times at 3283 NSP3CGAGUGGGCUGCUGCAGUUGAUGAA  93 1 times at 3286 NSP3GGGCUGCUGCAGUUGAUGAAGCGUU  94 1 times at 3291 NSP3GCAAGAAGAAGCACAACCAGUAGAA  95 1 times at 3352 NSP3CCAGUAGAAGUACCUGUUGAAGAUA  96 1 times at 3368 NSP3GCAGGUUGUCAUAGCUGACACCUUA  97 1 times at 3397 NSP3GGUUAUUACAGAGUGCGUUACCAUA  98 1 times at 3628 NSP3GGCGGUGGUAUCGCUGGUGCUAUUA  99 1 times at 3734 NSP3GCGGUGGUAUCGCUGGUGCUAUUAA 100 1 times at 3735 NSP3CGGUGGUAUCGCUGGUGCUAUUAAU 101 1 times at 3736 NSP3GCUGGUGCUAUUAAUGCGGCUUCAA 102 1 times at 3746 NSP3GCGGCUUCAAAAGGGGCUGUCCAAA 103 1 times at 3761 NSP3CGGCUUCAAAAGGGGCUGUCCAAAA 104 1 times at 3762 NSP3GGCUUCAAAAGGGGCUGUCCAAAAA 105 1 times at 3763 NSP3GCCGUUACAAGUAGGAGAUUCAGUU 106 1 times at 3817 NSP3CGUAGGCCCAGAUGCCCGCGCUAAA 107 1 times at 3883 NSP3CCCAGAUGCCCGCGCUAAACAGGAU 108 1 times at 3889 NSP3GGCUAUGAAUGCAUAUCCUCUUGUA 109 1 times at 3940 NSP3CCAGCUGUGUCUUUUGAUUAUCUUA 110 1 times at 4004 NSP3GCUGUGUCUUUUGAUUAUCUUAUUA 111 1 times at 4007 NSP3CUGUGUCUUUUGAUUAUCUUAUU  960 CGUCGUUAAUUCCCAAGAUGUCUAU 112 1 times at4057 NSP3 GGCGCAAUACGUAAAGCUAAAGAUU 113 1 times at 4142 NSP3GCGCAAUACGUAAAGCUAAAGAUUA 114 1 times at 4143 NSP3CGCAAUACGUAAAGCUAAAGAUUAU   1 1 times at 4144 NSP3CGCAAUACGUAAAGCUAAAGAUU  961 CGUAAAGCUAAAGAUUAUGGUUUUA 115 1 times at4151 NSP3 GCUAAAGAUUAUGGUUUUACUGUUU 116 1 times at 4157 NSP3GCACAGACAACUCUGCUAACACUAA 117 1 times at 4188 NSP3GGAACAAGGGUGUUGAUUAUACUAA 118 1 times at 4221 NSP3GGGUGUUGAUUAUACUAAGAAGUUU   2 1 times at 4228 NSP3GGGUGUUGAUUAUACUAAGAAGU  962 CGUCUAAGGACACUUUAGAUGAUAU 119 1 times at4287 NSP3 GGACACUUUAGAUGAUAUCUUACAA 120 1 times at 4294 NSP3GACACUUUAGAUGAUAUCUUACA  963 GCUAAUAAGUCUGUUGGUAUUAUAU 121 1 times at4322 NSP3 GGUAUUAUAUCUAUGCCUUUGGGAU 122 1 times at 4337 NSP3CCUUUGGGAUAUGUGUCUCAUGGUU 123 1 times at 4352 NSP3GCCCUACGUGUGUCUCCUAGCUAAU 124 1 times at 4420 NSP3CCCUACGUGUGUCUCCUAGCUAAUA 125 1 times at 4421 NSP3CCUACGUGUGUCUCCUAGCUAAUAA 126 1 times at 4422 NSP3GCUAAUAAAGAGCAAGAAGCUAUUU 127 1 times at 4439 NSP3GCAAGAAGCUAUUUUGAUGUCUGAA 128 1 times at 4450 NSP3GCUAUUUUGAUGUCUGAAGACGUUA 129 1 times at 4457 NSP3CGUUAAGUUAAACCCUUCAGAAGAU 130 1 times at 4477 NSP3CGUCCGCACUAAUGGUGGUUACAAU 131 1 times at 4513 NSP3CGCACUAAUGGUGGUUACAAUUCUU 132 1 times at 4517 NSP3CGCACUAAUGGUGGUUACAAUUC  964 CCUGCAUUGGUCUGAUCAAACCAUA 133 1 times at4594 NSP3 GGAUUCACGCACGACACAGCAGUUA 134 1 times at 4702 NSP3GCGUUUUCUUUAAUGGUGCUGAUAU 135 1 times at 4815 NSP3CGUUUUCUUUAAUGGUGCUGAUAUU 136 1 times at 4816 NSP3GCAGACAAUUUGACUGCUGAUGAAA 137 1 times at 4889 NSP3CCUACUUUCUUACACAGAUUCUAUU 138 1 times at 4949 NSP3UACUUUCUUACACAGAUUCUAUU  965 CGGUUACUUCAUACCGUGCUUGCAA 139 1 times at5222 NSP3 GGUUACUUCAUACCGUGCUUGCAAA 140 1 times at 5223 NSP3GCAUGGUUUGGAGAGAGUGGUGCAA 141 1 times at 5271 NSP3GCUUGUUGUUACGUGGGUGUGCAAA 142 1 times at 5336 NSP3CGUGGGUGUGCAAACUGUUGAAGAU 143 1 times at 5347 NSP3GGUUGCUGCUCUCAGGCACACCAAA 144 1 times at 5448 NSP3GCUGCUCUCAGGCACACCAAAUGAA 145 1 times at 5452 NSP3GCUCUCAGGCACACCAAAUGAAAAA 146 1 times at 5455 NSP3GGUGACAACCUCCACGGCGCCUGAU 147 1 times at 5482 NSP3GGGCAUUGAAACGGCUGUUGGCCAU 148 1 times at 5530 NSP3GGCAUUGAAACGGCUGUUGGCCAUU 149 1 times at 5531 NSP3GCAUUGAAACGGCUGUUGGCCAUUA 150 1 times at 5532 NSP3CCGUUAGCAAGACUUCAGACUGGAA 151 1 times at 5607 NSP3GCAAGACUUCAGACUGGAAGUGCAA 152 1 times at 5613 NSP3GGCCAAAAAUACAGUAGCGAUUGUA 153 1 times at 5660 NSP3GCCAAAAAUACAGUAGCGAUUGUAA 154 1 times at 5661 NSP3CCAAAAAUACAGUAGCGAUUGUAAU 155 1 times at 5662 NSP3CGUACGGUAUUCUUUGGACGGUAAU 156 1 times at 5689 NSP3GGACGGUAAUUUCAGAACAGAGGUU 157 1 times at 5704 NSP3CGGUAAUUUCAGAACAGAGGUUGAU 158 1 times at 5707 NSP3CCCGACCUAUCUGCUUUCUAUGUUA 159 1 times at 5732 NSP3CCGACCUAUCUGCUUUCUAUGUUAA 160 1 times at 5733 NSP3GACCUAUCUGCUUUCUAUGUUAA  966 CCUAUCUGCUUUCUAUGUUAAGGAU 161 1 times at5737 NSP3 GCUUUCUAUGUUAAGGAUGGUAAAU 162 1 times at 5744 NSP3GGAUGGUAAAUACUUUACAAGUGAA 163 1 times at 5758 NSP3CCACCCGUAACAUAUUCACCAGCUA 164 1 times at 5783 NSP3CCCGUAACAUAUUCACCAGCUACAA 165 1 times at 5786 NSP3CCGUAACAUAUUCACCAGCUACAAU 166 1 times at 5787 NSP3CGUAACAUAUUCACCAGCUACAAUU 167 1 times at 5788 NSP3GGACAACCUGGCGGUGAUGCUAUUA 168 1 times at 5858 NSP3GGCGGUGAUGCUAUUAGUUUGAGUU 169 1 times at 5867 NSP3GCGGUGAUGCUAUUAGUUUGAGUUU 170 1 times at 5868 NSP3CGGUGAUGCUAUUAGUUUGAGUUUU 171 1 times at 5869 NSP3GGUGAUGCUAUUAGUUUGAGUUUUA 172 1 times at 5870 NSP3GUGAUGCUAUUAGUUUGAGUUUU  967 CGGCGAUGUGUUGUUGGCUGAGUUU 173 1 times at5968 NSP3 GCUGAGUUUGACACUUAUGACCCUA 174 1 times at 5984 NSP3GGUGCCAUGUAUAAAGGCAAACCAA 175 1 times at 6020 NSP3GCAUCUUAUGAUACUAAUCUUAAUA 176 1 times at 6062 NSP3AUCUUAUGAUACUAAUCUUAAUA  968 CGUAGCCCCCAUUGAACUCGAAAAU 177 1 times at6121 NSP3 GCCCCCAUUGAACUCGAAAAUAAAU 178 1 times at 6125 NSP3CCCCAUUGAACUCGAAAAUAAAU  969 CCCCCAUUGAACUCGAAAAUAAAUU 179 1 times at6126 NSP3 CCCAUUGAACUCGAAAAUAAAUU  970 CCUUUCGUGAAGGACAAUGUCAGUU 180 1times at 6254 NSP3 CGUGAAGGACAAUGUCAGUUUCGUU 181 1 times at 6259 NSP3GGACAAUGUCAGUUUCGUUGCUGAU 182 1 times at 6265 NSP3CCCUAAGUAUCAAGUCAUUGUCUUA 183 1 times at 6352 NSP3CCUAAGUAUCAAGUCAUUGUCUUAA 184 1 times at 6353 NSP3CCCUAAGUAUCAAGUCAUUGUCU  971 GCACACCGUUGAGUCAGGUGAUAUU 185 1 times at6409 NSP3 CGUUGAGUCAGGUGAUAUUAACGUU 186 1 times at 6415 NSP3GGUGAUAUUAACGUUGUUGCAGCUU 187 1 times at 6425 NSP3GGGCUUCAUUUUAUUUCAAAGAAUU 188 1 times at 6486 NSP3GGCUUCAUUUUAUUUCAAAGAAUUU   4 1 times at 6487 NSP3GGCUUCAUUUUAUUUCAAAGAAU  972 GCUACCACUGCUGUAGGUAGUUGUA 189 1 times at6530 NSP3 CCACUGCUGUAGGUAGUUGUAUAAA 190 1 times at 6534 NSP3GGCAUAUUGACAGGCUGUUUUAGUU 191 1 times at 6590 NSP3GCAUAUUGACAGGCUGUUUUAGUUU 192 1 times at 6591 NSP3GCUUCCACUAGCUUACUUUAGUGAU 193 1 times at 6634 NSP3CCACUAGCUUACUUUAGUGAUUCAA 194 1 times at 6638 NSP3CACUAGCUUACUUUAGUGAUUCA  973 CCACAGAGGUUAAAGUGAGUGCUUU 195 1 times at6672 NSP3 GGCGUUGUGACAGGUAAUGUUGUAA 196 1 times at 6707 NSP3GCGUUGUGACAGGUAAUGUUGUAAA 197 1 times at 6708 NSP3CGUUGUGACAGGUAAUGUUGUAAAA 198 1 times at 6709 NSP3GCACUGCUGCUGUUGAUUUAAGUAU 199 1 times at 6741 NSP3GCUGCUGUUGAUUUAAGUAUGGAUA 200 1 times at 6746 NSP3CCGUGUGGAUUGGAAAUCAACCCUA 201 1 times at 6778 NSP3CGGUUGUUACUUAUGUUAUGCACAA 202 1 times at 6803 NSP3CCCAAGGUUUGAAAAAGUUCUACAA 203 1 times at 6906 NSP3CCCAAGGUUUGAAAAAGUUCUAC  974 CCAAGGUUUGAAAAAGUUCUACAAA 204 1 times at6907 NSP3 AAGGUUUGAAAAAGUUCUACAAA  975 GCUUGUGACGGUCUUGCUUCAGCUU 205 1times at 6962 NSP3 GCGCAAACCGUUCUGCAAUGUGUAA 206 1 times at 7020 NSP3CGCAAACCGUUCUGCAAUGUGUAAU 207 1 times at 7021 NSP3GCAAACCGUUCUGCAAUGUGUAAUU 208 1 times at 7022 NSP3GCAAUGUGUAAUUGGUGCUUGAUUA 209 1 times at 7034 NSP3GGUGCUUGAUUAGCCAAGAUUCCAU 210 1 times at 7047 NSP3CCAUAACUCACUACCCAGCUCUUAA 211 1 times at 7068 NSP3GGUUCAAACACAUCUUAGCCACUAU 212 1 times at 7096 NSP3GGCAGGUACAUUGCAUUAUUUCUUU 213 1 times at 7207 NSP3CAGGUACAUUGCAUUAUUUCUUU  976 CCAUAUUUGUAGACUGGCGGUCAUA 214 1 times at7242 NSP3 CGGUCAUACAAUUAUGCUGUGUCUA 215 1 times at 7259 NSP3GCUGUGUCUAGUGCCUUCUGGUUAU 216 1 times at 7274 NSP3GCUUUUACGCAAGUUUUAUCAGCAU 217 1 times at 7357 NSP3GCAAGUUUUAUCAGCAUGUAAUCAA 218 1 times at 7365 NSP3GCAUGUAAUCAAUGGUUGCAAAGAU 219 1 times at 7378 NSP3GCUCUGCUAUAAGAGGAACCGACUU 220 1 times at 7414 NSP3CGACUUACUAGAGUUGAAGCUUCUA 221 1 times at 7433 NSP3GCUUCUACCGUUGUCUGUGGUGGAA 222 1 times at 7451 NSP3CGGUAUUUCAUUCUGUCGUAGGCAU 223 1 times at 7504 NSP3GGUAUUUCAUUCUGUCGUAGGCAUA 224 1 times at 7505 NSP3GGGGAAUACCUUCAUCUGUGAAGAA 225 1 times at 7564 NSP3CCUUCAUCUGUGAAGAAGUCGCAAA 226 1 times at 7572 NSP3GCCCUACGCAGGCCUAUUAACGCUA 227 1 times at 7610 NSP3CGCAGGCCUAUUAACGCUACGGAUA 228 1 times at 7616 NSP3CGCUACGGAUAGAUCACAUUAUUAU 229 1 times at 7630 NSP3GGAUAGAUCACAUUAUUAUGUGGAU 230 1 times at 7636 NSP3CGUUACAGUUAAAGAGACUGUUGUU 231 1 times at 7663 NSP3CCUCUGCGCUUUUACAAAUCUAGAU 232 1 times at 7735 NSP3GCGCUUUUACAAAUCUAGAUAAGUU   5 1 times at 7740 NSP3GCGCUUUUACAAAUCUAGAUAAG  977 GGUCUGUAAAACUACUACUGGUAUA 233 1 times at7777 NSP3 GCUAGGUCUGCAUGUGUUUAUUAUU 234 1 times at 7856 NSP3GGUGAUUCUAGUGAAAUCGCCACUA 235 1 times at 7937 NSP3CGCCACUAAAAUGUUUGAUUCCUUU 236 1 times at 7954 NSP3CGCUGUAUAAUGUCACACGCGAUAA 237 1 times at 7995 NSP3CGUGAUGGCGUAAGGCGAGGCGAUA 238 1 times at 8045 NSP3CGUAAGGCGAGGCGAUAACUUCCAU 239 1 times at 8053 NSP3GGCGAUAACUUCCAUAGUGUCUUAA 240 1 times at 8063 NSP3CCAUAGUGUCUUAACAACAUUCAUU 241 1 times at 8074 NSP3CGGCUUCAGUUAACCAAAUUGUCUU 242 1 times at 8286 NSP3GGCUUCAGUUAACCAAAUUGUCU  978 CCAAAUUGUCUUGCGUAAUUCUAAU 243 1 times at8299 NSP3 CGACAGAUUCGCAUUGCAUGCCGUA 244 1 times at 8378 NSP3CGCAUUGCAUGCCGUAAGUGUAAUU   6 1 times at 8387 NSP3CGCAUUGCAUGCCGUAAGUGUAA  979 GCAUUGCAUGCCGUAAGUGUAAUUU 245 1 times at8388 NSP3 GCAUGCCGUAAGUGUAAUUUAGCUU 246 1 times at 8393 NSP3CCUCAAAGCUACGCGCUAAUGAUAA 247 1 times at 8430 NSP3CUCAAAGCUACGCGCUAAUGAUA  980 GCUACGCGCUAAUGAUAAUAUCUUA 248 1 times at8437 NSP3 CGCUAAUGAUAAUAUCUUAUCAGUU 249 1 times at 8443 NSP3GCUAAUGAUAAUAUCUUAUCAGUUA 250 1 times at 8444 NSP3CCGCAUCUUGGACUUUAAAGUUCUU 251 1 times at 8638 NSP4CCGCAUCUUGGACUUUAAAGUUC  981 CCUGAUGAUAAGUGCUUUGCUAAUA 252 1 times at8690 NSP4 GCUUUGCUAAUAAGCACCGGUCCUU 253 1 times at 8703 NSP4GCACCGGUCCUUCACACAAUGGUAU 254 1 times at 8716 NSP4CCGGUCCUUCACACAAUGGUAUCAU 255 1 times at 8719 NSP4GGUGCUCGCAUUCCAGACGUACCUA 256 1 times at 8816 NSP4GCUCGCAUUCCAGACGUACCUACUA 257 1 times at 8819 NSP4CGCAUUCCAGACGUACCUACUACAU 258 1 times at 8822 NSP4GCAUUCCAGACGUACCUACUACAUU 259 1 times at 8823 NSP4CCAGACGUACCUACUACAUUGGCUU 260 1 times at 8828 NSP4GCAUUCUUCCAUCUGAGUGCACUAU 261 1 times at 8964 NSP4GGGCCGUAUGACACCAUACUGCCAU 262 1 times at 9004 NSP4CCGUAUGACACCAUACUGCCAUGAU 263 1 times at 9007 NSP4CCAUACUGCCAUGAUCCUACUGUUU 264 1 times at 9017 NSP4GGCCUCAUGUUCGUUACGACUUGUA 265 1 times at 9072 NSP4GCCUCAUGUUCGUUACGACUUGUAU 266 1 times at 9073 NSP4CGACUUGUAUGAUGGUAACAUGUUU 267 1 times at 9088 NSP4CCACAAAUGGCUCGUGGGCCAUUUU 268 1 times at 9225 NSP4GGCCAUUUUUAAUGACCACCAUCUU 269 1 times at 9241 NSP4GCCAUUUUUAAUGACCACCAUCUUA 270 1 times at 9242 NSP4CCAUUUUUAAUGACCACCAUCUUAA 271 1 times at 9243 NSP4CCAUCUUAAUAGACCUGGUGUCUAU 272 1 times at 9259 NSP4CCUGGUGUCUAUUGUGGCUCUGAUU 273 1 times at 9272 NSP4GGUGUCUAUUGUGGCUCUGAUUUUA 274 1 times at 9275 NSP4GCAGUAUCACUGUUCCAGCCUAUUA 275 1 times at 9320 NSP4CCUAUUACUUAUUUCCAAUUGACUA 276 1 times at 9338 NSP4CCUCAUUGGUCUUGGGUAUAGGUUU 277 1 times at 9363 NSP4CCUGACUUUGCUCUUCUAUUAUAUU 278 1 times at 9397 NSP4GCUCUUCUAUUAUAUUAAUAAAGUA 279 1 times at 9406 NSP4CUCUUCUAUUAUAUUAAUAAAGU  982 GCUGUUGUUGCUGCUGUUCUUAAUA 280 1 times at9470 NSP4 CCUGCAUUUAUUAUGCAUGUUUCUU 281 1 times at 9587 NSP4CCAGGACGCUGCCUCUAAUAUCUUU 282 1 times at 9760 NSP4GGACGCUGCCUCUAAUAUCUUUGUU 283 1 times at 9763 NSP4CGCUGCCUCUAAUAUCUUUGUUAUU 284 1 times at 9766 NSP4GCUGCCUCUAAUAUCUUUGUUAUUA 285 1 times at 9767 NSP4UGCCUCUAAUAUCUUUGUUAUUA  983 CCUCUAAUAUCUUUGUUAUUAACAA 286 1 times at9771 NSP4 CUCUAAUAUCUUUGUUAUUAACA  984 GCAGCUCUUAGAAACUCUUUAACUA 287 1times at 9806 NSP4 CAGCUCUUAGAAACUCUUUAACU  985CCUAUUCACGAUUUUUGGGGUUGUU 288 1 times at 9837 NSP4GGUUGUUUAACAAGUAUAAGUACUU 289 1 times at 9855 NSP4GCCGCUUAUCGUGAAGCUGCAGCAU 290 1 times at 9899 NSP4GCGAGACUGGUAGUGAUCUUCUUUA 291 1 times at 9954 NSP4CCUCUGGCGUGUUGCAAAGCGGUUU 292 1 times at 10002 NSP4GCGUGUUGCAAAGCGGUUUGGUGAA 293 1 times at 10008 NSP4CGUGUUGCAAAGCGGUUUGGUGAAA 294 1 times at 10009 NSP4GGUUACCUGCGGUAGCAUGACUCUU 295 1 times at 10075 NSP5CGGUAGCAUGACUCUUAAUGGUCUU 296 1 times at 10084 NSP5GGUAGCAUGACUCUUAAUGGUCUUU 297 1 times at 10085 NSP5CCUAAUUAUGAUGCCUUGUUGAUUU 298 1 times at 10172 NSP5CGCUCCAGCAAACUUGCGUGUUGUU 299 1 times at 10237 NSP5GGUCAUGCCAUGCAAGGCACUCUUU 300 1 times at 10262 NSP5GGCGCAGCAUUUAGUGUGUUAGCAU 301 1 times at 10352 NSP5GCAUUUAGUGUGUUAGCAUGCUAUA 302 1 times at 10358 NSP5CCGACUGGUACAUUCACUGUUGUAA 303 1 times at 10391 NSP5CGACUGGUACAUUCACUGUUGUAAU 304 1 times at 10392 NSP5CGCCCUAACUACACAAUUAAGGGUU 305 1 times at 10418 NSP5CCGGUUCAGCAUUUGAUGGUACUAU 306 1 times at 10545 NSP5GCACCAAGUUCAGUUAACAGACAAA 307 1 times at 10597 NSP5GCUUGGCUUUACGCAGCAAUACUUA 308 1 times at 10643 NSP5GCAGCAAUACUUAAUGGUUGCGCUU 309 1 times at 10655 NSP5GGCGUUGCUAUUGAACAGCUGCUUU 310 1 times at 10793 NSP5GCGUUGCUAUUGAACAGCUGCUUUA 311 1 times at 10794 NSP5CGUUGCUAUUGAACAGCUGCUUUAU 312 1 times at 10795 NSP5GGAAGAUGAAUUCACACCUGAGGAU 313 1 times at 10879 NSP5CCUGAGGAUGUUAAUAUGCAGAUUA 314 1 times at 10895 NSP5GGUUAUGCAGAGUGGUGUGAGAAAA 315 1 times at 10927 NSP5GGUGUGAGAAAAGUUACAUAUGGUA 316 1 times at 10940 NSP6CGACCCUUGUCUCAACCUAUGUGAU 317 1 times at 10983 NSP6CCCUUGUCUCAACCUAUGUGAUAAU 318 1 times at 10986 NSP6CCACUAAAUUUACUUUGUGGAACUA 319 1 times at 11019 NSP6CCCACACAGUUGUUCCCACUCUUAU 320 1 times at 11060 NSP6CCACACAGUUGUUCCCACUCUUAUU 321 1 times at 11061 NSP6GGCCUUCGUUAUGUUGUUGGUUAAA 322 1 times at 11095 NSP6CGUUAUGUUGUUGGUUAAACACAAA 323 1 times at 11101 NSP6GCCUGUGGCUAUUUGUUUGACUUAU 324 1 times at 11152 NSP6GCAAACAUAGUCUACGAGCCCACUA 325 1 times at 11177 NSP6CGUCAGCGCUGAUUGCAGUUGCAAA 326 1 times at 11211 NSP6GCUGAUUGCAGUUGCAAAUUGGCUU 327 1 times at 11218 NSP6GGCUUGCCCCCACUAAUGCUUAUAU 328 1 times at 11238 NSP6CCCACUAAUGCUUAUAUGCGCACUA 329 1 times at 11246 NSP6GGUGUAAUGUGGUUGUACACUUAUA 330 1 times at 11378 NSP6GCAUUGGAGAAGCCUCAAGCCCCAU 331 1 times at 11403 NSP6CCGGAAGUGAAGAUGAUACUUUUAU 332 1 times at 11555 NSP6CGGAAGUGAAGAUGAUACUUUUAUU 333 1 times at 11556 NSP6CGGAAGUGAAGAUGAUACUUUUA  986 GGAAGUGAAGAUGAUACUUUUAUUA 334 1 times at11557 NSP6 AAGUGAAGAUGAUACUUUUAUUA  987 GCUUAGAGCACCUAUGGGUGUCUAU 335 1times at 11644 NSP6 GCACCUAUGGGUGUCUAUGACUUUA 336 1 times at 11651 NSP6GCUAACAAUCUAACUGCACCUAGAA 337 1 times at 11708 NSP6GCACCUAGAAAUUCUUGGGAGGCUA 338 1 times at 11723 NSP6GGGAGGCUAUGGCUCUGAACUUUAA 339 1 times at 11739 NSP6GGUUGCUGCUAUGCAGUCUAAACUU 340 1 times at 11797 NSP6GCAGUCUAAACUUACAGAUCUUAAA 341 1 times at 11809 NSP6CCAACAGUUACACUUAGAGGCUAAU 342 1 times at 11863 NSP7GGGCUUUCUGUGUUAAAUGCCAUAA 343 1 times at 11898 NSP7GGCUUUCUGUGUUAAAUGCCAUAAU 344 1 times at 11899 NSP7GCAGCAACAGACCCCAGUGAGGCUU 345 1 times at 11933 NSP7GCUAGUGAUAUUUUUGACACUCCUA 346 1 times at 12026 NSP7CCUAGCGUACUUCAAGCUACUCUUU 347 1 times at 12047 NSP7GCGCAGAAAGCCUAUCAGGAAGCUA 348 1 times at 12113 NSP8CGCAGAAAGCCUAUCAGGAAGCUAU 349 1 times at 12114 NSP8GGACUCUGGUGACACCUCACCACAA 350 1 times at 12139 NSP8GGUGACACCUCACCACAAGUUCUUA 351 1 times at 12146 NSP8CCUCACCACAAGUUCUUAAGGCUUU 352 1 times at 12153 NSP8GGCUUUGCAGAAGGCUGUUAAUAUA 353 1 times at 12172 NSP8GCAGAAGGCUGUUAAUAUAGCUAAA 354 1 times at 12178 NSP8GCUAAAAACGCCUAUGAGAAGGAUA 355 1 times at 12197 NSP8GGAUAAGGCAGUGGCCCGUAAGUUA 356 1 times at 12217 NSP8GCAGUGGCCCGUAAGUUAGAACGUA 357 1 times at 12224 NSP8GGCUAUGACUUCUAUGUAUAAGCAA 358 1 times at 12259 NSP8GGCUAUGACUUCUAUGUAUAAGC  988 GCAAAAAUUGUCAGUGCUAUGCAAA 359 1 times at12305 NSP8 GCUAUGCAAACUAUGUUGUUUGGUA 360 1 times at 12320 NSP8GCAAACUAUGUUGUUUGGUAUGAUU 361 1 times at 12325 NSP8GCUUCAAAUAAACUUCGCGUUGUAA 362 1 times at 12434 NSP8CCGUCUGGAAUCAGGUAGUCACAUA 363 1 times at 12471 NSP8CGUCUGGAAUCAGGUAGUCACAUAU 364 1 times at 12472 NSP8CCCUCGCUUAACUACGCUGGGGCUU 365 1 times at 12497 NSP8CCUCGCUUAACUACGCUGGGGCUUU 366 1 times at 12498 NSP8GGGGCUUUGUGGGACAUUACAGUUA 367 1 times at 12515 NSP8GGGCUUUGUGGGACAUUACAGUUAU 368 1 times at 12516 NSP8GGCUUUGUGGGACAUUACAGUUAUA 369 1 times at 12517 NSP8GCUUUGUGGGACAUUACAGUUAUAA 370 1 times at 12518 NSP8GGGCAUCCACUUCUGCCGUUAAGUU 371 1 times at 12630 NSP8CCACUUCUGCCGUUAAGUUGCAAAA 372 1 times at 12636 NSP8CCGUUAAGUUGCAAAAUAAUGAGAU 373 1 times at 12645 NSP8GGUCAAGAGCAAACUAACUGUAAUA 374 1 times at 12707 NSP9GGGUCGUAAAAUGCUGAUGGCUCUU 375 1 times at 12763 NSP9CGUAAAAUGCUGAUGGCUCUUCUUU 376 1 times at 12767 NSP9GCUGAUGGCUCUUCUUUCUGAUAAU 377 1 times at 12775 NSP9GGCUCUUCUUUCUGAUAAUGCCUAU 378 1 times at 12781 NSP9GCGCGUGUUGAAGGUAAGGACGGAU 379 1 times at 12815 NSP9CGCGUGUUGAAGGUAAGGACGGAUU 380 1 times at 12816 NSP9GCGUGUUGAAGGUAAGGACGGAUUU 381 1 times at 12817 NSP9GCAAAUUCUUGAUUGCGGGACCAAA 382 1 times at 12867 NSP9GGACCAAAAGGACCUGAAAUCCGAU 383 1 times at 12884 NSP9GGGCACAUUGCUGCGACUGUUAGAU 384 1 times at 12959 NSP9GGCACAUUGCUGCGACUGUUAGAUU 385 1 times at 12960 NSP9GCGACUGUUAGAUUGCAAGCUGGUU 386 1 times at 12971 NSP9GCAAGCUGGUUCUAACACCGAGUUU 387 1 times at 12985 NSP9GGUUCUAACACCGAGUUUGCCUCUA 388 1 times at 12992 NSP10CCUAAAACUGGUACAGGUAUAGCUA 389 1 times at 13127 NSP10GGUACAGGUAUAGCUAUAUCUGUUA 390 1 times at 13136 NSP10UACAGGUAUAGCUAUAUCUGUUA  989 GCUAUAUCUGUUAAACCAGAGAGUA 391 1 times at13148 NSP10 CCGUGCGCAUAUAGAACAUCCUGAU 392 1 times at 13219 NSP10CCUGUAAUGUCUGUCAAUAUUGGAU 393 1 times at 13335 NSP10GCCCCAAUCUAAAGAUUCCAAUUUU 394 1 times at 13402 NSP10CCCCAAUCUAAAGAUUCCAAUUUUU 395 1 times at 13403 NSP10CCCCAAUCUAAAGAUUCCAAUUU  990 CCCAAUCUAAAGAUUCCAAUUUUUU 396 1 times at13404 NSP10 CCCAAUCUAAAGAUUCCAAUUUU  991 CCAAUCUAAAGAUUCCAAUUUUUUA 397 1times at 13405 NSP10 CGGGGUUCUAUUGUAAAUGCCCGAA 398 1 times at 13438NSP12 GGGGUUCUAUUGUAAAUGCCCGAAU 399 1 times at 13439 NSP12GGGUUCUAUUGUAAAUGCCCGAAUA 400 1 times at 13440 NSP12CGAAUAGAACCCUGUUCAAGUGGUU 401 1 times at 13459 NSP12GGGCAUUUGACAUCUGCAACUAUAA 402 1 times at 13505 NSP12GGCUAAGGUUGCUGGUAUUGGAAAA 403 1 times at 13530 NSP12GCUAAGGUUGCUGGUAUUGGAAAAU 404 1 times at 13531 NSP12GGUAUUGGAAAAUACUACAAGACUA 405 1 times at 13543 NSP12GGAAAAUACUACAAGACUAAUACUU 406 1 times at 13549 NSP12CCAAGGGCAUCAUUUAGACUCCUAU 407 1 times at 13596 NSP12CGUUAAGAGGCAUACUAUGGAGAAU 408 1 times at 13626 NSP12GCAUACUAUGGAGAAUUAUGAACUA 409 1 times at 13635 NSP12CCAUGAUUUCUUCAUCUUUGAUGUA 410 1 times at 13707 NSP12CCUCAUAUUGUACGUCAGCGUUUAA 411 1 times at 13747 NSP12CGUCAGCGUUUAACUGAGUACACUA 412 1 times at 13759 NSP12GCCCUGAGGCACUUUGAUCAAAAUA 413 1 times at 13801 NSP12GCUUAAGGCUAUCUUAGUGAAGUAU 414 1 times at 13833 NSP12GCUGUGAUGUUACCUACUUUGAAAA 415 1 times at 13862 NSP12CUGUGAUGUUACCUACUUUGAAA  992 CCUACUUUGAAAAUAAACUCUGGUU 416 1 times at13874 NSP12 CCCAGUGUUAUUGGUGUUUAUCAUA   7 1 times at 13915 NSP12CCCAGUGUUAUUGGUGUUUAUCA  993 CCAGUGUUAUUGGUGUUUAUCAUAA 417 1 times at13916 NSP12 CAGUGUUAUUGGUGUUUAUCAUA  994 CGCCAAGCUAUCUUAAACACUGUUA 418 1times at 13957 NSP12 GCCAAGCUAUCUUAAACACUGUUAA 419 1 times at 13958NSP12 CCAAGCUAUCUUAAACACUGUUAAA 420 1 times at 13959 NSP12GCUAUCUUAAACACUGUUAAAUUUU 421 1 times at 13963 NSP12GCUCACACUAGACAACCAGGACCUU 422 1 times at 14022 NSP12CCAGGACCUUAAUGGCAAGUGGUAU 423 1 times at 14037 NSP12GGACCUUAAUGGCAAGUGGUAUGAU 424 1 times at 14040 NSP12CCUUAAUGGCAAGUGGUAUGAUUUU 425 1 times at 14043 NSP12GCAAGUGGUAUGAUUUUGGUGACUU 426 1 times at 14051 NSP12GGUAUGAUUUUGGUGACUUCGUAAU 427 1 times at 14057 NSP12GGUUCAGGAGUAGCUAUAGUUGAUA 428 1 times at 14092 NSP12GCUAUAGUUGAUAGCUACUAUUCUU 429 1 times at 14104 NSP12CGAUUGUCUGGCCGCUGAGACACAU 430 1 times at 14154 NSP12CGCUGAGACACAUAGGGAUUGUGAU 431 1 times at 14166 NSP12GCUGAGACACAUAGGGAUUGUGAUU 432 1 times at 14167 NSP12GGUACAACUCUUUGAGAAGUACUUU 433 1 times at 14247 NSP12UACAACUCUUUGAGAAGUACUUU  995 CGCAAAUUGCGUUAAUUGUACUGAU 434 1 times at14295 NSP12 CCGUUGUGUGUUACAUUGUGCUAAU 435 1 times at 14322 NSP12CGUUGUGUGUUACAUUGUGCUAAUU 436 1 times at 14323 NSP12GCUAAUUUCAAUGUAUUGUUUGCUA 437 1 times at 14341 NSP12GCCUAAGACUUGUUUCGGACCCAUA 438 1 times at 14373 NSP12CGGACCCAUAGUCCGAAAGAUCUUU 439 1 times at 14388 NSP12GCCAUUUGUAGUAUCUUGUGGUUAU 440 1 times at 14424 NSP12GGUUAUCACUACAAAGAAUUAGGUU 441 1 times at 14443 NSP12GGUUUAGUCAUGAAUAUGGAUGUUA 442 1 times at 14464 NSP12CCAGCCAUGCACAUUGCCUCCUCUA 443 1 times at 14542 NSP12GCACAUUGCCUCCUCUAACGCUUUU 444 1 times at 14550 NSP12GCCUCCUCUAACGCUUUUCUUGAUU 445 1 times at 14557 NSP12CCUCCUCUAACGCUUUUCUUGAUUU 446 1 times at 14558 NSP12CUCCUCUAACGCUUUUCUUGAUU 996 GCUUUUCUUGAUUUGAGGACAUCAU 447 1 times at14569 NSP12 GCUGCACUUACAACUGGUUUGACUU 448 1 times at 14605 NSP12GGCCUGGCAAUUUUAACCAAGACUU 449 1 times at 14642 NSP12CCAAGACUUCUAUGAUUUCGUGGUA 450 1 times at 14658 NSP12GCUCAAACAUUUUUUCUUUGCUCAA 451 1 times at 14718 NSP12GCUCAAGAUGGUAAUGCUGCUAUUA 452 1 times at 14737 NSP12GGUAAUGCUGCUAUUACAGAUUAUA 453 1 times at 14746 NSP12GCUAUUACAGAUUAUAAUUACUAUU 454 1 times at 14755 NSP12GCCUACUAUGUGUGACAUCAAACAA 455 1 times at 14790 NSP12CCUACUAUGUGUGACAUCAAACAAA 456 1 times at 14791 NSP12UACUAUGUGUGACAUCAAACAAA  997 GCAUGGAAGUUGUAAACAAGUACUU 457 1 times at14825 NSP12 GGAAGUUGUAAACAAGUACUUCGAA 458 1 times at 14829 NSP12CGAAAUCUAUGACGGUGGUUGUCUU 459 1 times at 14850 NSP12CGGUGGUUGUCUUAAUGCUUCUGAA 460 1 times at 14862 NSP12GCUUCUGAAGUGGUUGUUAAUAAUU 461 1 times at 14878 NSP12GCCAUCCUUUUAAUAAGUUUGGCAA 462 1 times at 14918 NSP12CCAUCCUUUUAAUAAGUUUGGCAAA 463 1 times at 14919 NSP12CGUGUCUAUUAUGAGAGCAUGUCUU 464 1 times at 14947 NSP12GCAGGCGUGUCCAUACUUAGCACAA 465 1 times at 15082 NSP12CGCCAGUACCAUCAGAAAAUGCUUA 466 1 times at 15115 NSP12GCCAGUACCAUCAGAAAAUGCUUAA 467 1 times at 15116 NSP12CGUGGAGCGACUUGCGUCAUUGGUA 468 1 times at 15157 NSP12GGAGCGACUUGCGUCAUUGGUACUA 469 1 times at 15160 NSP12GCGACUUGCGUCAUUGGUACUACAA 470 1 times at 15163 NSP12CGACUUGCGUCAUUGGUACUACAAA 471 1 times at 15164 NSP12GCGUCAUUGGUACUACAAAGUUCUA 472 1 times at 15170 NSP12GGUGGCUGGGAUUUCAUGCUUAAAA 473 1 times at 15196 NSP12GGCUGGGAUUUCAUGCUUAAAACAU 474 1 times at 15199 NSP12GCUGGGAUUUCAUGCUUAAAACAUU 475 1 times at 15200 NSP12UGGGAUUUCAUGCUUAAAACAUU  998 GGGAUUUCAUGCUUAAAACAUUGUA   8 1 times at15203 NSP12 GGGAUUUCAUGCUUAAAACAUUG  999 GGGUUGGGAUUACCCUAAGUGUGAU 476 1times at 15255 NSP12 GGUUGGGAUUACCCUAAGUGUGAUA 477 1 times at 15256NSP12 CCUAAGUGUGAUAGAGCUAUGCCUA 478 1 times at 15268 NSP12CCUAAUAUGUGUAGAAUCUUCGCUU 479 1 times at 15289 NSP12CGCUUCACUCAUAUUAGCUCGUAAA 480 1 times at 15309 NSP12GGGACAGAUUUUAUCGCUUGGCAAA 481 1 times at 15356 NSP12GGACAGAUUUUAUCGCUUGGCAAAU 482 1 times at 15357 NSP12GGCAAAUGAGUGUGCUCAGGUGCUA 483 1 times at 15375 NSP12GCAAAUGAGUGUGCUCAGGUGCUAA 484 1 times at 15376 NSP12GGUUACUACGUCAAACCUGGAGGUA 485 1 times at 15424 NSP12CCACUGCAUAUGCCAAUAGUGUCUU 486 1 times at 15467 NSP12CACUGCAUAUGCCAAUAGUGUCU 1000 GGGUGCUAAUGGCAACAAGAUUGUU   9 1 times at15534 NSP12 GGGUGCUAAUGGCAACAAGAUUG 1001 GGAGCACUAGCCCAGACCCCAAAUU 487 1times at 15608 NSP12 GCCCAGACCCCAAAUUUGUUGAUAA 488 1 times at 15617NSP12 CCCAGACCCCAAAUUUGUUGAUAAA 489 1 times at 15618 NSP12CCAGACCCCAAAUUUGUUGAUAAAU 490 1 times at 15619 NSP12CCCCAAAUUUGUUGAUAAAUACUAU  10 1 times at 15624 NSP12CCCCAAAUUUGUUGAUAAAUACU 1002 GCUUUUCUUAAUAAGCACUUUUCUA 491 1 times at15649 NSP12 CGGUGUCGUUUGCUAUAAUAGUGAU 492 1 times at 15693 NSP12GGUGUCGUUUGCUAUAAUAGUGAUU 493 1 times at 15694 NSP12GCUAUAAUAGUGAUUAUGCAGCUAA 494 1 times at 15704 NSP12GCAGCUAAGGGUUACAUUGCUGGAA 495 1 times at 15721 NSP12GGGUUACAUUGCUGGAAUACAGAAU 496 1 times at 15729 NSP12GGUUACAUUGCUGGAAUACAGAAUU 497 1 times at 15730 NSP12GGAAACGCUGUAUUAUCAGAACAAU 498 1 times at 15759 NSP12CGCUGUAUUAUCAGAACAAUGUCUU 499 1 times at 15764 NSP12GCUGUAUUAUCAGAACAAUGUCUUU 500 1 times at 15765 NSP12GCUGGGUGGAAACCGAUCUGAAGAA 501 1 times at 15806 NSP12CGAUCUGAAGAAAGGGCCACAUGAA 502 1 times at 15819 NSP12GCCACAUGAAUUCUGUUCACAGCAU 503 1 times at 15834 NSP12CCACAUGAAUUCUGUUCACAGCAUA 504 1 times at 15835 NSP12GCUUUAUAUUAAGGAUGGCGACGAU 505 1 times at 15861 NSP12GGAUGGCGACGAUGGUUACUUCCUU 506 1 times at 15873 NSP12GGCGACGAUGGUUACUUCCUUCCUU 507 1 times at 15877 NSP12GCGACGAUGGUUACUUCCUUCCUUA 508 1 times at 15878 NSP12CGACGAUGGUUACUUCCUUCCUUAU 509 1 times at 15879 NSP12CCUUAUCCAGACCCUUCAAGAAUUU 510 1 times at 15898 NSP12CCUUCAAGAAUUUUGUCUGCCGGUU 511 1 times at 15910 NSP12CGGUUGCUUUGUAGAUGAUAUCGUU  11 1 times at 15930 NSP12CGGUUGCUUUGUAGAUGAUAUCG 1003 GGUUGCUUUGUAGAUGAUAUCGUUA 512 1 times at15931 NSP12 UUGCUUUGUAGAUGAUAUCGUUA 1004 GCGGUUUGUGUCUUUGGCUAUAGAU 513 1times at 15981 NSP12 GCUAUAGAUGCUUACCCUCUCACAA 514 1 times at 15997NSP12 CCCUCUCACAAAGCAUGAAGAUAUA 515 1 times at 16011 NSP12CUCUCACAAAGCAUGAAGAUAUA 1005 GCAUGAAGAUAUAGAAUACCAGAAU 516 1 times at16023 NSP12 CCAGAAUGUAUUCUGGGUCUACUUA 517 1 times at 16041 NSP12GGGUCUACUUACAGUAUAUAGAAAA 518 1 times at 16055 NSP12GGUCUACUUACAGUAUAUAGAAAAA 519 1 times at 16056 NSP12GUCUACUUACAGUAUAUAGAAAA 1006 GCUUGACAGUUAUUCUGUCAUGCUA 520 1 times at16107 NSP12 CCUACCACUUUGCAGGCUGUCGGUU 521 1 times at 16192 NSP12GCAGGCUGUCGGUUCAUGCGUUGUA 522 1 times at 16203 NSP12CCACAUAAGAUGGUUUUGUCUGUUU 523 1 times at 16318 NSP13CCACUUUGCGCUAAUGGUCUUGUAU 524 1 times at 16450 NSP13GCGCUAAUGGUCUUGUAUUCGGCUU 525 1 times at 16457 NSP13CGCUAAUGGUCUUGUAUUCGGCUUA 526 1 times at 16458 NSP13GCUAAUGGUCUUGUAUUCGGCUUAU 527 1 times at 16459 NSP13GGUGAUUACACCCUUGCCAAUACUA 528 1 times at 16558 NSP13CCAAUACUACAACAGAACCACUCAA 529 1 times at 16574 NSP13CCACCACUCAAUCGUAAUUAUGUUU 530 1 times at 16726 NSP13ACCACUCAAUCGUAAUUAUGUUU 1007 CCACUCAAUCGUAAUUAUGUUUUUA 531 1 times at16729 NSP13 GGUUAUCAUAUAACCAAAAAUAGUA 532 1 times at 16756 NSP13GCGCAUUGAUUAUAGUGAUGCUGUA 533 1 times at 16809 NSP13CGCAUUGAUUAUAGUGAUGCUGUAU 534 1 times at 16810 NSP13GCUGUAUCCUACAAGUCUAGUACAA 535 1 times at 16828 NSP13CCUACAAGUCUAGUACAACGUAUAA 536 1 times at 16835 NSP13UACAAGUCUAGUACAACGUAUAA 1008 CGUAUAAACUGACUGUAGGUGACAU 537 1 times at16853 NSP13 GGCUACCUUGACGGCGCCCACAAUU 538 1 times at 16902 NSP13GGUAUGUUAAAAUUACUGGGUUGUA 539 1 times at 16940 NSP13GCCAACUUCCAAAAAUCAGGUUAUA 540 1 times at 17005 NSP13CCAAAAAUCAGGUUAUAGUAAAUAU 541 1 times at 17013 NSP13GCACGUGUUGUUUAUACAGCAUGUU 542 1 times at 17110 NSP13CGCAGCUGUUGAUGCUUUGUGUGAA 543 1 times at 17139 NSP13GCAGCUGUUGAUGCUUUGUGUGAAA 544 1 times at 17140 NSP13GCUUUGUGUGAAAAAGCUUUUAAAU 545 1 times at 17152 NSP13GCUUUUAAAUAUUUGAACAUUGCUA 546 1 times at 17167 NSP13CGUGUUGAGUGCUAUGACAGGUUUA 547 1 times at 17221 NSP13GGUUAGUAUGUGCACUAAUUAUGAU 548 1 times at 17331 NSP13GCACUAAUUAUGAUCUUUCAAUUAU 549 1 times at 17342 NSP13CACUAAUUAUGAUCUUUCAAUUA 1009 GCACAGUUGCCAGCUCCUAGGACUU 550 1 times at17413 NSP13 CCAGCUCCUAGGACUUUGUUGACUA 551 1 times at 17422 NSP13GGACUUUGUUGACUAGAGGCACAUU 552 1 times at 17432 NSP13GCACUGUGAGCGCUCUUGUCUACAA 553 1 times at 17555 NSP13GCGCUCUUGUCUACAAUAAUAAAUU 554 1 times at 17564 NSP13GCUUUAAAAUACUCUAUAAGGGCAA 555 1 times at 17618 NSP13CGCAUGAUGCUAGCUCUGCCAUUAA 556 1 times at 17648 NSP13GCAUGAUGCUAGCUCUGCCAUUAAU 557 1 times at 17649 NSP13GCCAUUAAUAGACCACAACUCACAU 558 1 times at 17665 NSP13CCAUUAAUAGACCACAACUCACAUU 559 1 times at 17666 NSP13CCACAACUCACAUUUGUGAAGAAUU 560 1 times at 17677 NSP13CCGGCAUGGAGUAAGGCAGUCUUUA 561 1 times at 17716 NSP13CGGCAUGGAGUAAGGCAGUCUUUAU 562 1 times at 17717 NSP13GGCAUGGAGUAAGGCAGUCUUUAUU 563 1 times at 17718 NSP13GCAUGGAGUAAGGCAGUCUUUAUUU 564 1 times at 17719 NSP13AUGGAGUAAGGCAGUCUUUAUUU 1010 CCUCACAGGGUUCAGAAUACCAGUA 565 1 times at17810 NSP13 GCACAUGCUAACAACAUUAACAGAU 566 1 times at 17863 NSP13CACAUGCUAACAACAUUAACAGA 1011 GCAAUCACUCGUGCCCAAAAAGGUA 567 1 times at17896 NSP13 GCCCAAAAAGGUAUUCUUUGUGUUA 568 1 times at 17908 NSP13GCCCAAAAAGGUAUUCUUUGUGU 1012 CCCAAAAAGGUAUUCUUUGUGUUAU 569 1 times at17909 NSP13 GGCACUCUUUGAGUCCUUAGAGUUU 570 1 times at 17943 NSP13GCACUCUUUGAGUCCUUAGAGUUUA 571 1 times at 17944 NSP13CACUCUUUGAGUCCUUAGAGUUU 1013 CCUUAGAGUUUACUGAAUUGUCUUU 572 1 times at17957 NSP13 CCUUUUUAAAGAUUGCUCUAGAGAA 573 1 times at 18018 NSP14GGCCUCUCACCUGCUUAUGCACCAA 574 1 times at 18049 NSP14GCGUGAAUCUUAAUUUACCCGCAAA 575 1 times at 18119 NSP14CGUGAAUCUUAAUUUACCCGCAAAU 576 1 times at 18120 NSP14CGCAAAUGUCCCAUACUCUCGUGUU 577 1 times at 18138 NSP14GCAAAUGUCCCAUACUCUCGUGUUA 578 1 times at 18139 NSP14CGUGUUAUUUCCAGGAUGGGCUUUA 579 1 times at 18157 NSP14GGGCUUUAAACUCGAUGCAACAGUU 580 1 times at 18174 NSP14GGCAAGUUCGAAGCUGGAUAGGCUU 581 1 times at 18242 NSP14GGUGCUCAUGCUUCCCGUAAUGCAU 582 1 times at 18277 NSP14CCAAUGUGCCUCUACAAUUAGGAUU 583 1 times at 18308 NSP14GGUGUUGUAGACACUGAGUGGGGUA 584 1 times at 18367 NSP14CGUCCUCCACCAGGUGAACAGUUUA 585 1 times at 18415 NSP14CGUUUGUUUGUUGGGCUCAUGGCUU 586 1 times at 18545 NSP14GGCUUUGAAUUAACGUCUGCAUCAU 587 1 times at 18565 NSP14GCUUUGAAUUAACGUCUGCAUCAUA 588 1 times at 18566 NSP14CGUCUGCAUCAUACUUUUGCAAGAU 589 1 times at 18578 NSP14GCAUCAUACUUUUGCAAGAUAGGUA 590 1 times at 18583 NSP14GCAGCGUACUCUUCACCUCUGCAAU 591 1 times at 18643 NSP14GCGUACUCUUCACCUCUGCAAUCUU 592 1 times at 18646 NSP14CGUACUCUUCACCUCUGCAAUCUUA 593 1 times at 18647 NSP14GCAAUCUUAUGCCUGCUGGACUCAU 594 1 times at 18663 NSP14GCCUGCUGGACUCAUUCCUGCGGUU 595 1 times at 18673 NSP14CCUGCUGGACUCAUUCCUGCGGUUA 596 1 times at 18674 NSP14GGACUCAUUCCUGCGGUUAUGAUUA 597 1 times at 18680 NSP14CCUGCGGUUAUGAUUAUGUCUACAA 598 1 times at 18689 NSP14CUGCGGUUAUGAUUAUGUCUACA 1014 GGUUAUGAUUAUGUCUACAACCCUU 599 1 times at18694 NSP14 CGAUGUUCAACAGUGGGGUUAUGUA 600 1 times at 18726 NSP14CGAUCGUUAUUGCUCUGUCCAUCAA 601 1 times at 18771 NSP14GCUCAUGUGGCUUCUAAUGAUGCAA 602 1 times at 18799 NSP14GCAAUAAUGACUCGUUGUUUAGCUA 603 1 times at 18820 NSP14CGUUGUUUAGCUAUUCAUUCUUGUU 604 1 times at 18832 NSP14CCUUAUAUCUCACAUGAAAAGAAAU 605 1 times at 18889 NSP14GCGCAACGUCGUACGUGCUGCUCUU 606 1 times at 18939 NSP14CGGUUCAUUUGACAAAGUCUAUGAU 607 1 times at 18969 NSP14CGGUUCAUUUGACAAAGUCUAUG 1015 GGUUCAUUUGACAAAGUCUAUGAUA 608 1 times at18970 NSP14 UUCAUUUGACAAAGUCUAUGAUA 1016 GGCAUUAUUUUGAUGCACAGCCCUU 609 1times at 19043 NSP14 GGACAUGGCCUCAAGAUUUGCUGAU 610 1 times at 19101NSP14 GCACGCUUUUCAUACACCAGCAUAU 611 1 times at 19257 NSP14CGCUUUUCAUACACCAGCAUAUGAU 612 1 times at 19260 NSP14CCUUUACCAUUCUUUUAUUAUUCUA 613 1 times at 19309 NSP14GGUAAUGGUAGUAUGAUAGAGGAUA 614 1 times at 19354 NSP14GGUAGUAUGAUAGAGGAUAUUGAUU 615 1 times at 19360 NSP14UAGUAUGAUAGAGGAUAUUGAUU 1017 GGAUAUUGAUUAUGUACCCCUAAAA 616 1 times at19374 NSP14 CCCCUAAAAUCUGCAGUCUGUAUUA 617 1 times at 19390 NSP14GGUGUUAUAAGACCUUUGAUAUUUA 618 1 times at 19517 NSP14GUGUUAUAAGACCUUUGAUAUUU 1018 CCAUUUUAUUGGUGUUGAGGGUGAA 619 1 times at19611 NSP15 CCACUUUGCCUACUAAUAUAGCUUU 620 1 times at 19712 NSP15GCGUGCUGUACGCUCGCAUCCCGAU 621 1 times at 19752 NSP15CGUGCUGUACGCUCGCAUCCCGAUU 622 1 times at 19753 NSP15CCCGAUUUCAAAUUGCUACACAAUU 623 1 times at 19771 NSP15CCGAUUUCAAAUUGCUACACAAUUU 624 1 times at 19772 NSP15CGAUUUCAAAUUGCUACACAAUUUA 625 1 times at 19773 NSP15GCUACACAAUUUACAAGCAGACAUU 626 1 times at 19785 NSP15GCUACAAGUUCGUCCUUUGGGAUUA 627 1 times at 19811 NSP15CCUUUGGGAUUAUGAACGUAGCAAU 628 1 times at 19824 NSP15GGGAUUAUGAACGUAGCAAUAUUUA 629 1 times at 19829 NSP15GGGAUUAUGAACGUAGCAAUAUU 1019 GGAUUAUGAACGUAGCAAUAUUUAU 630 1 times at19830 NSP15 CGUAGCAAUAUUUAUGGUACUGCUA 631 1 times at 19840 NSP15GCAAUAUUUAUGGUACUGCUACUAU 632 1 times at 19844 NSP15CCCAAUGCCAUCUUUAUUUCUGAUA 633 1 times at 19966 NSP15GCCAUCUUUAUUUCUGAUAGAAAAA 634 1 times at 19972 NSP15GCCAUCUUUAUUUCUGAUAGAAA 1020 CCAUCUUUAUUUCUGAUAGAAAAAU 635 1 times at19973 NSP15 AUCUUUAUUUCUGAUAGAAAAAU 1021 CCCUUGUAUGGUAGGUCCUGAUUAU 636 1times at 20007 NSP15 CCGUGAUAGUGAUGUUGUUAAACAA 637 1 times at 20055NSP15 CCGUGAUAGUGAUGUUGUUAAAC 1022 GGAAAACUAUGCUUUUGAGCACGUA 638 1 timesat 20244 NSP15 CGUUAGGCGGUCUUCACUUGCUUAU 639 1 times at 20294 NSP15GGCGGUCUUCACUUGCUUAUUGGUU 640 1 times at 20299 NSP15GCGGUCUUCACUUGCUUAUUGGUUU 641 1 times at 20300 NSP15CGGUCUUCACUUGCUUAUUGGUUUA 642 1 times at 20301 NSP15GGUCUUCACUUGCUUAUUGGUUUAU 643 1 times at 20302 NSP15GUCUUCACUUGCUUAUUGGUUUA 1023 GCUUAUUGGUUUAUACAAGAAGCAA 644 1 times at20313 NSP15 GGAAGGUCAUAUUAUUAUGGAAGAA 645 1 times at 20340 NSP15GCUAAAAGGUAGCUCAACUAUUCAU 646 1 times at 20367 NSP15GGUAGCUCAACUAUUCAUAACUAUU 647 1 times at 20374 NSP15GCUCAACUAUUCAUAACUAUUUUAU 648 1 times at 20378 NSP15CUCAACUAUUCAUAACUAUUUUA 1024 GGCUUUUAAGGCGGUGUGUUCUGUU 649 1 times at20421 NSP15 GCUUUUAAGGCGGUGUGUUCUGUUA 650 1 times at 20422 NSP15GGCGGUGUGUUCUGUUAUAGAUUUA 651 1 times at 20430 NSP15GCGGUGUGUUCUGUUAUAGAUUUAA 652 1 times at 20431 NSP15CGGUGUGUUCUGUUAUAGAUUUAAA 653 1 times at 20432 NSP15GCUUGACGACUUUGUUAUGAUUUUA 654 1 times at 20457 NSP15CGUAGUAUCCAAGGUUGUCAAGGUU 655 1 times at 20499 NSP15GGUUGUCAAGGUUCCUAUUGACUUA 656 1 times at 20511 NSP15GGUUCCUAUUGACUUAACAAUGAUU 657 1 times at 20520 NSP15UUCCUAUUGACUUAACAAUGAUU 1025 CCCUCGACUCCAGGCUUCUGCAGAU 658 1 times at20589 NSP15 CCUCGACUCCAGGCUUCUGCAGAUU 659 1 times at 20590 NSP15GCCAUCCCUCUUUAAAGUUCAAAAU 660 1 times at 20634 NSP16CCCUCUUUAAAGUUCAAAAUGUAAA 661 1 times at 20639 NSP16CUCUUUAAAGUUCAAAAUGUAAA 1026 CGCGGUGUGCACAUGAACAUCGCUA 662 1 times at20713 NSP16 GCGGUGUGCACAUGAACAUCGCUAA 663 1 times at 20714 NSP16CGGUGUGCACAUGAACAUCGCUAAA 664 1 times at 20715 NSP16GGUGUGCACAUGAACAUCGCUAAAU 665 1 times at 20716 NSP16GCCAGUAUUUAAAUACUUGCACAUU 666 1 times at 20753 NSP16CCAGUAUUUAAAUACUUGCACAUUA 667 1 times at 20754 NSP16GCCUGCCAAUAUGCGUGUUAUACAU 668 1 times at 20784 NSP16CCUGCCAAUAUGCGUGUUAUACAUU 669 1 times at 20785 NSP16UGCCAAUAUGCGUGUUAUACAUU 1027 CGUGUUAUACAUUUUGGCGCUGGUU 670 1 times at20797 NSP16 GCCAUUAUUAUAGAUAAUGAUUUAA 671 1 times at 20878 NSP16CCAUUAUUAUAGAUAAUGAUUUAAA 672 1 times at 20879 NSP16CGUGUCAGAUGCUGACAUAACUUUA 673 1 times at 20910 NSP16GCUGACAUAACUUUAUUUGGAGAUU 674 1 times at 20920 NSP16CCGACAUGUAUGAUCCUACUACUAA 675 1 times at 20987 NSP16GACAUGUAUGAUCCUACUACUAA 1028 CCUACUACUAAGAAUGUAACAGGUA 676 1 times at21001 NSP16 GGUAGUAAUGAGUCAAAGGCUUUAU 677 1 times at 21022 NSP16GCUUUAUUCUUUACUUACCUGUGUA 678 1 times at 21040 NSP16CCUGUGUAACCUCAUUAAUAAUAAU 679 1 times at 21057 NSP16GGUGGGUCUGUUGCUAUUAAAAUAA 680 1 times at 21091 NSP16GCUAUUAAAAUAACAGAACACUCUU 681 1 times at 21103 NSP16GGAGCGUUGAACUUUAUGAACUUAU 682 1 times at 21128 NSP16GAGCGUUGAACUUUAUGAACUUA 1029 GGGAAAAUUUGCUUGGUGGACUGUU 683 1 times at21153 NSP16 GGAAAAUUUGCUUGGUGGACUGUUU 684 1 times at 21154 NSP16GCAAAUGCAUCCUCAUCUGAAGGAU 685 1 times at 21190 NSP16GGUAUUAAUUACUUGGGUACUAUUA 686 1 times at 21223 NSP16GGGUACUAUUAAAGAAAAUAUAGAU 687 1 times at 21237 NSP16GGGUACUAUUAAAGAAAAUAUAG 1030 GGUGGUGCUAUGCACGCCAACUAUA 688 1 times at21262 NSP16 GGUGCUAUGCACGCCAACUAUAUAU 689 1 times at 21265 NSP16GCUAUGCACGCCAACUAUAUAUUUU 690 1 times at 21268 NSP16CGCCAACUAUAUAUUUUGGAGAAAU 691 1 times at 21276 NSP16GCCAACUAUAUAUUUUGGAGAAAUU 692 1 times at 21277 NSP16CCACUCCUAUGAAUCUGAGUACUUA 693 1 times at 21302 NSP16GGAGAGUCAAAUUAACGAACUCGUA 694 1 times at 21390 NSP16GGGUAAGUUACUUAUCCGUGACAAU 695 1 times at 21432 NSP16CCGUGACAAUGAUACACUCAGUGUU 696 1 times at 21447 NSP16CGUGACAAUGAUACACUCAGUGUUU 697 1 times at 21448 NSP16GGCUGACGGUAUUAUAUACCCUCAA 698 1 times at 21610 S proteinGGUAUUAUAUACCCUCAAGGCCGUA 699 1 times at 21617 S proteinGGCCGUACAUAUUCUAACAUAACUA  12 1 times at 21635 S proteinGGCCGUACAUAUUCUAACAUAAC 1031 GCCGUACAUAUUCUAACAUAACUAU 700 1 times at21636 S protein GCCGUACAUAUUCUAACAUAACU 1032 CCCUAUCAGGGAGACCAUGGUGAUA701 1 times at 21680 S protein CCUAUCAGGGAGACCAUGGUGAUAU 702 1 times at21681 S protein GGGAGACCAUGGUGAUAUGUAUGUU 703 1 times at 21688 S proteinCACUUUACUUAGAGCUUUUUAUU 1033 GGAGACCAUGGUGAUAUGUAUGUUU 704 1 times at21689 S protein CCAUCUACCAGCGCUACUAUACGAA 705 1 times at 21854 S proteinCCAGCGCUACUAUACGAAAAAUUUA 706 1 times at 21861 S proteinGGGCCGCUUCUUCAAUCAUACUCUA 707 1 times at 21937 S proteinGCCCGAUGGAUGUGGCACUUUACUU 708 1 times at 21970 S proteinCCCGAUGGAUGUGGCACUUUACUUA 709 1 times at 21971 S proteinGGAUGUGGCACUUUACUUAGAGCUU 710 1 times at 21977 S proteinGGCACUUUACUUAGAGCUUUUUAUU 711 1 times at 21983 S proteinCCUGCUGGCAAUUCCUAUACUUCUU 712 1 times at 22040 S proteinGCAACAGAUUGUUCUGAUGGCAAUU 713 1 times at 22085 S proteinCGUAAUGCCAGUCUGAACUCUUUUA 714 1 times at 22115 S proteinCCAGUCUGAACUCUUUUAAGGAGUA 715 1 times at 22122 S proteinCGUAACUGCACCUUUAUGUACACUU 716 1 times at 22157 S proteinGCACCUUUAUGUACACUUAUAACAU 717 1 times at 22164 S proteinCACCUUUAUGUACACUUAUAACA 1034 CCGAAGAUGAGAUUUUAGAGUGGUU  13 1 times at22191 S protein CCGAAGAUGAGAUUUUAGAGUGG 1035 CGAAGAUGAGAUUUUAGAGUGGUUU718 1 times at 22192 S protein GCUCAAGGUGUUCACCUCUUCUCAU 719 1 times at22232 S protein CCUCUUCUCAUCUCGGUAUGUUGAU 720 1 times at 22246 S proteinGGUAUGUUGAUUUGUACGGCGGCAA 721 1 times at 22260 S proteinCCGUUAACUUUCCUGUUGGAUUUUU 722 1 times at 22412 S proteinGGAUUUUUCUGUUGAUGGUUAUAUA 723 1 times at 22429 S proteinCGCAGAGCUAUAGACUGUGGUUUUA 724 1 times at 22454 S proteinGCAGAGCUAUAGACUGUGGUUUUAA 725 1 times at 22455 S proteinGCUAUAGACUGUGGUUUUAAUGAUU 726 1 times at 22460 S proteinCCACUGCUCAUAUGAAUCCUUCGAU 727 1 times at 22495 S proteinCCUUCGAUGUUGAAUCUGGAGUUUA 728 1 times at 22512 S proteinCGAAGCAAAACCUUCUGGCUCAGUU 729 1 times at 22552 S proteinGGCUGAAGGUGUUGAAUGUGAUUUU 730 1 times at 22585 S proteinGCUGAAGGUGUUGAAUGUGAUUUUU 731 1 times at 22586 S proteinGGCACACCUCCUCAGGUUUAUAAUU 732 1 times at 22625 S proteinGCACACCUCCUCAGGUUUAUAAUUU 733 1 times at 22626 S proteinCCUCAGGUUUAUAAUUUCAAGCGUU 734 1 times at 22634 S proteinGGUUUAUAAUUUCAAGCGUUUGGUU 735 1 times at 22639 S proteinGCGUUUGGUUUUUACCAAUUGCAAU 736 1 times at 22654 S proteinCGUUUGGUUUUUACCAAUUGCAAUU 737 1 times at 22655 S proteinGGUUUUUACCAAUUGCAAUUAUAAU 738 1 times at 22660 S proteinGCUUUCACUUUUUUCUGUGAAUGAU 739 1 times at 22696 S proteinGCUGGUCCAAUAUCCCAGUUUAAUU 740 1 times at 22835 S proteinGGUCCAAUAUCCCAGUUUAAUUAUA 741 1 times at 22838 S proteinUGGUCCAAUAUCCCAGUUUAAUU 1036 CCCAGUUUAAUUAUAAACAGUCCUU  14 1 times at22848 S protein CCCAGUUUAAUUAUAAACAGUCC 1037 CCAGUUUAAUUAUAAACAGUCCUUU742 1 times at 22849 S protein CCUUUUCUAAUCCCACAUGUUUGAU 743 1 times at22869 S protein CCUUACUACUAUUACUAAGCCUCUU 744 1 times at 22915 S proteinCCUCAGUUAGUGAACGCUAAUCAAU 745 1 times at 22997 S proteinCGCUAAUCAAUACUCACCCUGUGUA 746 1 times at 23011 S proteinGCUAAUCAAUACUCACCCUGUGUAU 747 1 times at 23012 S proteinGGGAAGACGGUGAUUAUUAUAGGAA 748 1 times at 23058 S proteinGGAAGACGGUGAUUAUUAUAGGAAA 749 1 times at 23059 S proteinCGGUGAUUAUUAUAGGAAACAACUA 750 1 times at 23065 S proteinGGUGAUUAUUAUAGGAAACAACUAU 751 1 times at 23066 S proteinGGCUGGCUUGUUGCUAGUGGCUCAA 752 1 times at 23108 S proteinGCUUGUUGCUAGUGGCUCAACUGUU 753 1 times at 23113 S proteinGCAAUUACAGAUGGGCUUUGGUAUU 754 1 times at 23149 S proteinGGGCUUUGGUAUUACAGUUCAAUAU 755 1 times at 23161 S proteinGCUUGAAUUUGCUAAUGACACAAAA 756 1 times at 23215 S proteinGCAAUUGCGUGGAAUAUUCCCUCUA 757 1 times at 23256 S proteinCGUGGAAUAUUCCCUCUAUGGUGUU 758 1 times at 23263 S proteinGGUGUUCGACAGCAGCGCUUUGUUU 759 1 times at 23324 S proteinGCUAUUAUUCUGAUGAUGGCAACUA 760 1 times at 23373 S proteinCCCGUUCUACGCGAUCAAUGCUUAA 761 1 times at 23523 S proteinGGUUGUGUCCUAGGACUUGUUAAUU 762 1 times at 23588 S proteinCCUCUUUGUUCGUAGAGGACUGCAA 763 1 times at 23613 S proteinGCGCUUGGCAUCCAUUGCUUUUAAU 764 1 times at 23725 S proteinGGUUGAUCAACUUAAUAGUAGUUAU 765 1 times at 23761 S proteinUUGAUCAACUUAAUAGUAGUUAU 1038 CCUUUGGUGUGACUCAGGAGUACAU 766 1 times at23814 S protein CCAUGGUGCCAAUUUACGCCAGGAU 767 1 times at 23959 S proteinGGUGCCAAUUUACGCCAGGAUGAUU 768 1 times at 23963 S proteinCGCCAGGAUGAUUCUGUACGUAAUU 769 1 times at 23975 S proteinGCCAGGAUGAUUCUGUACGUAAUUU 770 1 times at 23976 S proteinCAGGAUGAUUCUGUACGUAAUUU 1039 GGAUGAUUCUGUACGUAAUUUGUUU 771 1 times at23980 S protein AUGAUUCUGUACGUAAUUUGUUU 1040 CGUAAUUUGUUUGCGAGCGUGAAAA772 1 times at 23993 S protein GCGAGCGUGAAAAGCUCUCAAUCAU 773 1 times at24005 S protein CCAGGUUUUGGAGGUGACUUUAAUU 774 1 times at 24041 S proteinCAGGUUUUGGAGGUGACUUUAAU 1041 GGCAGUCGUAGUGCACGUAGUGCUA 775 1 times at24098 S protein GCAGUCGUAGUGCACGUAGUGCUAU 776 1 times at 24099 S proteinCGUAGUGCUAUUGAGGAUUUGCUAU 777 1 times at 24113 S proteinGCUGAUCCUGGUUAUAUGCAAGGUU 778 1 times at 24155 S proteinGGUUAUAUGCAAGGUUACGAUGAUU 779 1 times at 24164 S proteinGGUCCAGCAUCAGCUCGUGAUCUUA 780 1 times at 24200 S proteinCCAGCAUCAGCUCGUGAUCUUAUUU 781 1 times at 24203 S proteinGCUCGUGAUCUUAUUUGUGCUCAAU 782 1 times at 24212 S proteinGGAUGUUAAUAUGGAAGCCGCGUAU 783 1 times at 24271 S proteinGGUGUUGGCUGGACUGCUGGCUUAU 784 1 times at 24323 S proteinGCUGGACUGCUGGCUUAUCCUCCUU 785 1 times at 24330 S proteinGCUGGCUUAUCCUCCUUUGCUGCUA 786 1 times at 24338 S proteinGCUGCUAUUCCAUUUGCACAGAGUA 787 1 times at 24356 S proteinCGGUGUUGGCAUUACUCAACAGGUU 788 1 times at 24397 S proteinGGUUCUUUCAGAGAACCAAAAGCUU 789 1 times at 24418 S proteinCCAAAAGCUUAUUGCCAAUAAGUUU 790 1 times at 24433 S proteinGGAGCUAUGCAAACAGGCUUCACUA 791 1 times at 24470 S proteinGCUAUGCAAACAGGCUUCACUACAA 792 1 times at 24473 S proteinGCAAACAGGCUUCACUACAACUAAU 793 1 times at 24478 S proteinGGCUUCACUACAACUAAUGAAGCUU  15 1 times at 24485 S proteinGGCUUCACUACAACUAAUGAAGC 1042 GCUUCACUACAACUAAUGAAGCUUU 794 1 times at24486 S protein GCUAUCUAAUACUUUUGGUGCUAUU 795 1 times at 24571 S proteinGGCACAAUCCAAGCGUUCUGGAUUU 796 1 times at 24778 S proteinGCACAAUCCAAGCGUUCUGGAUUUU 797 1 times at 24779 S proteinCCCUAGCAACCACAUUGAGGUUGUU 798 1 times at 24880 S proteinCCUAGCAACCACAUUGAGGUUGUUU 799 1 times at 24881 S proteinCCACAUUGAGGUUGUUUCUGCUUAU 800 1 times at 24889 S proteinCCCUACUAAUUGUAUAGCCCCUGUU 801 1 times at 24934 S proteinCCUACUAAUUGUAUAGCCCCUGUUA 802 1 times at 24935 S proteinGCCCCUGUUAAUGGCUACUUUAUUA 803 1 times at 24950 S proteinCCCCUGUUAAUGGCUACUUUAUUAA  16 1 times at 24951 S proteinCCCCUGUUAAUGGCUACUUUAUU 1043 CCCUGUUAAUGGCUACUUUAUUAAA  17 1 times at24952 S protein CCCUGUUAAUGGCUACUUUAUUA 1044 CCUGUUAAUGGCUACUUUAUUAAAA804 1 times at 24953 S protein GGUCAUAUACUGGCUCGUCCUUCUA 805 1 times at25005 S protein CCUUAAUGAGUCUUACAUAGACCUU 806 1 times at 25279 S proteinGGCAAUUAUACUUAUUACAACAAAU 807 1 times at 25313 S proteinGGCCGUGGUACAUUUGGCUUGGUUU 808 1 times at 25338 S proteinGCUGGGCUUGUUGCCUUAGCUCUAU 809 1 times at 25367 S proteinGCACUGGUUGUGGCACAAACUGUAU 810 1 times at 25413 S proteinGGUUGUGGCACAAACUGUAUGGGAA 811 1 times at 25418 S proteinGGCACAAACUGUAUGGGAAAACUUA 812 1 times at 25424 S proteinGCACAAACUGUAUGGGAAAACUUAA 813 1 times at 25425 S proteinCACAAACUGUAUGGGAAAACUUA 1045 GGAAAACUUAAGUGUAAUCGUUGUU 814 1 times at25439 S protein CGUUGUUGUGAUAGAUACGAGGAAU 815 1 times at 25457 S proteinGCCGCAUAAGGUUCAUGUUCACUAA  18 1 times at 25492 S proteinGCCGCAUAAGGUUCAUGUUCACU 1046 CCGCAUAAGGUUCAUGUUCACUAAU 816 1 times at25493 S protein CGCAUAAGGUUCAUGUUCACUAAUU 817 1 times at 25494 S proteinGCAUAAGGUUCAUGUUCACUAAUUA 818 1 times at 25495 S proteinGGUUGCAUGCUUAGGGCUUGUAUUA 819 1 times at 25639 orf 3CCAAGCUGAUACAGCUGGUCUUUAU 820 1 times at 25671 orf 3GCUGAUACAGCUGGUCUUUAUACAA 821 1 times at 25675 orf 3CGAAUUGACGUCCCAUCUGCAGAAU 822 1 times at 25705 orf 3CCCUGUGCUGUGGAACUGUCAGCUA 823 1 times at 25973 orf4aCCUGUGCUGUGGAACUGUCAGCUAU 824 1 times at 25974 orf4aGCUGUGGAACUGUCAGCUAUCCUUU 825 1 times at 25979 orf4aGCUAUCCUUUGCUGGUUAUACUGAA 826 1 times at 25994 orf4aGCUGGUUAUACUGAAUCUGCUGUUA 827 1 times at 26004 orf4aGGUUAUACUGAAUCUGCUGUUAAUU 828 1 times at 26007 orf4aGCCAAACAGGACGCAGCUCAGCGAA 829 1 times at 26046 orf4aCCAAACAGGACGCAGCUCAGCGAAU 830 1 times at 26047 orf4aGGUUGCUACAUAAGGAUGGAGGAAU 831 1 times at 26077 orf4aCGGCACUCAAGUUUAUUCGCGCAAA 832 1 times at 26127 orf4aCCAACACACUAUGUCAGGGUUACAU 833 1 times at 26248 orf4bGGGUUACAUUUUCAGACCCCAACAU 834 1 times at 26264 orf4bGGUAUCUACGUUCGGGUCAUCAUUU 835 1 times at 26291 orf4bGCCAACCUGUUUCUGAGUACCAUAU 836 1 times at 26351 orf4bCCAACCUGUUUCUGAGUACCAUAUU 837 1 times at 26352 orf4bCCAUAUUACUCUAGCUUUGCUAAAU 838 1 times at 26370 orf4bGCUAAAUCUCACUGAUGAAGAUUUA 839 1 times at 26388 orf4bCGCCUUGCUGCGCAAAACUCUUGUU 840 1 times at 26475 orf4bGCUGCGCAAAACUCUUGUUCUUAAU 841 1 times at 26481 orf4bCUGCGCAAAACUCUUGUUCUUAA 1047 CGCAAAACUCUUGUUCUUAAUGCAU 842 1 times at26485 orf4b CGCAAAACUCUUGUUCUUAAUGC 1048 GGAUUGGCUUCUCGUUCAGGGAUUU 843 1times at 26583 orf4b GCUUCUCGUUCAGGGAUUUUCCCUU 844 1 times at 26589orf4b CGUUCAGGGAUUUUCCCUUUACCAU 845 1 times at 26595 orf4bCCCUUUACCAUAGUGGCCUCCCUUU 846 1 times at 26609 orf4bCCUUUACCAUAGUGGCCUCCCUUUA 847 1 times at 26610 orf4bCGCAAUUACAUCAUUACAAUGCCAU 848 1 times at 26677 orf4bCCUCAACAAAUGUUUGUUACUCCUU 849 1 times at 26716 orf4bCCAUACGGUCUUCCAAUCAGGGUAA 850 1 times at 26759 orf4bGGUAAUAAACAAAUUGUUCAUUCUU 851 1 times at 26779 orf4bGGCUUUCUCGGCGUCUUUAUUUAAA 852 1 times at 26841 orf5GGCUUUCUCGGCGUCUUUAUUUA 1049 CCUAUUAUUACUGCUACGUCAAGAU 853 1 times at26991 orf5 CCUUGUUCUGUAUAACUUUUUAUUA 854 1 times at 27057 orf5UUGUUCUGUAUAACUUUUUAUUA 1050 GGUGUACAUUAUCCAACUGGAAGUU 855 1 times at27100 orf5 CCUCAUAAUACUUUGGUUUGUAGAU 856 1 times at 27147 orf5CCAAACCAUUAUUUAUUAGAAACUU 857 1 times at 27284 orf5GCGUUGCAGCUGUUCUCGUUGUUUU 858 1 times at 27315 orf5CGUUGCAGCUGUUCUCGUUGUUUUU 859 1 times at 27316 orf5GCAGCUGUUCUCGUUGUUUUUAUUU 860 1 times at 27320 orf5CCACUUAUAUAGAGUGCACUUAUAU 861 1 times at 27353 orf5GCACUUAUAUUAGCCGUUUUAGUAA 862 1 times at 27368 orf5CCGUUUUAGUAAGAUUAGCCUAGUU 863 1 times at 27381 orf5CGUUUUAGUAAGAUUAGCCUAGUUU 864 1 times at 27382 orf5CGCGCGAUUCAGUUCCUCUUCACAU 865 1 times at 27461 orf5GCGCGAUUCAGUUCCUCUUCACAUA 866 1 times at 27462 orf5CGCGAUUCAGUUCCUCUUCACAUAA 867 1 times at 27463 orf5GCGAUUCAGUUCCUCUUCACAUAAU 868 1 times at 27464 orf5CGCCCCGAGCUCGCUUAUCGUUUAA 869 1 times at 27489 orf5CGUUUAAGCAGCUCUGCGCUACUAU 870 1 times at 27507 orf5GGGUCCCGUGUAGAGGCUAAUCCAU 871 1 times at 27532 GGUCCCGUGUAGAGGCUAAUCCAUU872 1 times at 27533 GGACAUAUGGAAAACGAACUAUGUU 873 1 times at 27569GACAUAUGGAAAACGAACUAUGU 1051 CCGUAGUAUGUGCUAUAACACUCUU 874 1 times at27647 E GGCUUUCCUUACGGCUACUAGAUUA 875 1 times at 27681 EGCUUUCCUUACGGCUACUAGAUUAU 876 1 times at 27682 EGCUACUAGAUUAUGUGUGCAAUGUA 877 1 times at 27694 ECCCUGUUAGUUCAGCCCGCAUUAUA 878 1 times at 27734 ECCCAUCCCGUAGUAUGACUGUCUAU 879 1 times at 27965 MGGCCAUCUUCCAUGGCGCUAUCAAU 880 1 times at 28021 MGCCAUCUUCCAUGGCGCUAUCAAUA 881 1 times at 28022 MCCAUCUUCCAUGGCGCUAUCAAUAU 882 1 times at 28023 MCCAAUUGAUCUAGCUUCCCAGAUAA 883 1 times at 28062 MGGCAUUGUAGCAGCUGUUUCAGCUA 887 1 times at 28092 M GGCAUUGUAGCAGCUGUUUCAGC1052 GCAUUGUAGCAGCUGUUUCAGCUAU 885 1 times at 28093 MGCUGUUUCAGCUAUGAUGUGGAUUU 886 1 times at 28104 MGGAUUUCCUACUUUGUGCAGAGUAU 887 1 times at 28123 MCGGCUGUUUAUGAGAACUGGAUCAU 888 1 times at 28149 MCCAGUGUAACUGCUGUUGUAACCAA 889 1 times at 28261 MCCACCUCAAAAUGGCUGGCAUGCAU 890 1 times at 28289 MGCAUGCAUUUCGGUGCUUGUGACUA 891 1 times at 28306 MCGGUGCUUGUGACUACGACAGACUU 892 1 times at 28316 MGCUUGUGACUACGACAGACUUCCUA 893 1 times at 28320 MGCUUUAAAAAUGGUGAAGCGGCAAA 894 1 times at 28380 MGGAACUAAUUCCGGCGUUGCCAUUU 895 1 times at 28410 MCCGGCGUUGCCAUUUACCAUAGAUA 896 1 times at 28420 MCGGCGUUGCCAUUUACCAUAGAUAU 897 1 times at 28421 MGGCGUUGCCAUUUACCAUAGAUAUA 898 1 times at 28422 MGCGUUGCCAUUUACCAUAGAUAUAA 899 1 times at 28423 MGCAGGUAAUUACAGGAGUCCGCCUA 900 1 times at 28449 MGGUAAUUACAGGAGUCCGCCUAUUA 901 1 times at 28452 MGGAGUCCGCCUAUUACGGCGGAUAU 902 1 times at 28462 MGCCUAUUACGGCGGAUAUUGAACUU 903 1 times at 28469 M GCCUAUUACGGCGGAUAUUGAAC1053 GGCGGAUAUUGAACUUGCAUUGCUU 904 1 times at 28478 MGCAUUGCUUCGAGCUUAGGCUCUUU 905 1 times at 28494 MGCUUCGAGCUUAGGCUCUUUAGUAA 906 1 times at 28499 MGGCAGGGUGUACCUCUUAAUGCCAA 907 1 times at 28743 NGCAGGGUGUACCUCUUAAUGCCAAU 908 1 times at 28744 NGGGUAUUGGCGGAGACAGGACAGAA 909 1 times at 28790 NGGUAUUGGCGGAGACAGGACAGAAA 910 1 times at 28791 NGGCGGAGACAGGACAGAAAAAUUAA 911 1 times at 28797 NGCGGAGACAGGACAGAAAAAUUAAU 912 1 times at 28798 NCGGAGACAGGACAGAAAAAUUAAUA 913 1 times at 28799 NGGACAGAAAAAUUAAUACCGGGAAU 914 1 times at 28807 NGCAGCACUCCCAUUCCGGGCUGUUA 915 1 times at 28889 NCCGGGCUGUUAAGGAUGGCAUCGUU 916 1 times at 28903 NCGGGCUGUUAAGGAUGGCAUCGUUU 917 1 times at 28904 NGGAUGGCAUCGUUUGGGUCCAUGAA 918 1 times at 28915 NGGCGCCACUGAUGCUCCUUCAACUU 919 1 times at 28943 NGCGCCACUGAUGCUCCUUCAACUUU 920 1 times at 28944 NCGCCACUGAUGCUCCUUCAACUUUU 921 1 times at 28945 NGGGACGCGGAACCCUAACAAUGAUU 922 1 times at 28970 NCCGGUACUAAGCUUCCUAAAAACUU 923 1 times at 29019 N CCGGUACUAAGCUUCCUAAAAAC1054 CCACAUUGAGGGGACUGGAGGCAAU 924 1 times at 29044 NGGGACUGGAGGCAAUAGUCAAUCAU 925 1 times at 29054 NGGAGGCAAUAGUCAAUCAUCUUCAA 926 1 times at 29060 N GAGGCAAUAGUCAAUCAUCUUCA1055 CGGAGCAGUAGGAGGUGAUCUACUU 927 1 times at 29182 NGGAGCAGUAGGAGGUGAUCUACUUU 928 1 times at 29183 NCCUUGAUCUUCUGAACAGACUACAA 929 1 times at 29209 NGGCAAAGUAAAGCAAUCGCAGCCAA 930 1 times at 29246 NGCAAAGUAAAGCAAUCGCAGCCAAA 931 1 times at 29247 NCGCAGCCAAAAGUAAUCACUAAGAA 932 1 times at 29262 NGCGCCACAAGCGCACUUCCACCAAA 933 1 times at 29314 NCGCCACAAGCGCACUUCCACCAAAA 934 1 times at 29315 NGCACUUCCACCAAAAGUUUCAACAU 935 1 times at 29325 NCGCGGACCAGGAGACCUCCAGGGAA 936 1 times at 29369 NGCGGACCAGGAGACCUCCAGGGAAA 937 1 times at 29370 NCCUCCAGGGAAACUUUGGUGAUCUU 938 1 times at 29383 NCCAGGGAAACUUUGGUGAUCUUCAA 939 1 times at 29386 NCCCCAAAUUGCUGAGCUUGCUCCUA 940 1 times at 29444 NGCUUGCUCCUACAGCCAGUGCUUUU 941 1 times at 29458 NCCUACAGCCAGUGCUUUUAUGGGUA 942 1 times at 29465 NGCUUUUAUGGGUAUGUCGCAAUUUA 943 1 times at 29477 NCGCAAUUUAAACUUACCCAUCAGAA 944 1 times at 29493 NGCAACCCUGUGUACUUCCUUCGGUA 945 1 times at 29532 NCCUUCGGUACAGUGGAGCCAUUAAA 946 1 times at 29548 NGGUUGGAGCUUCUUGAGCAAAAUAU 947 1 times at 29604 NGGAGCUUCUUGAGCAAAAUAUUGAU 948 1 times at 29608 N GAGCUUCUUGAGCAAAAUAUUGA1056 GGAAAAGAAACAAAAGGCACCAAAA 949 1 times at 29656 NCGUCCAAGUGUUCAGCCUGGUCCAA 950 1 times at 29759 NCCAAUGAUUGAUGUUAACACUGAUU 951 1 times at 29780 N

TABLE 2 Predicted 25 mer siRNA targeting MERS NC019843.325mer blunt ended sequences SEQ ID Start Protein23 mer Sequences passing all SEQ ID SiRNA sequence NO: Base Namemetrics and BLAST search NO: CCCAGAAUCUGCUUAAGAAGUUGAU  32 1 times at825 NSP1 CCCAGAAUCUGCUUAAGAAGUUG  952 GCCCAUUCAUGGAUAAUGCUAUUAA  64 1times at 1884 NSP2 GCCCAUUCAUGGAUAAUGCUAUU  953CCCAUUCAUGGAUAAUGCUAUUAAU  65 1 times at 1885 NSP2CCCAUUCAUGGAUAAUGCUAUUA  954 CGCCAUUACUGCACCUUAUGUAGUU  67 1 times at1936 NSP2 CGCCAUUACUGCACCUUAUGUAG  955 GGCGACUUUAUGUCUACAAUUAUUA  71 1times at 2186 NSP2 GGCGACUUUAUGUCUACAAUUAU  957GCUGUGUCUUUUGAUUAUCUUAUUA 111 1 times at 4007 NSP3CUGUGUCUUUUGAUUAUCUUAUU  960 CGCAAUACGUAAAGCUAAAGAUUAU   1 1 times at4144 NSP3 CGCAAUACGUAAAGCUAAAGAUU  961 GGGUGUUGAUUAUACUAAGAAGUUU   2 1times at 4228 NSP3 GGGUGUUGAUUAUACUAAGAAGU  962GGACACUUUAGAUGAUAUCUUACAA 120 1 times at 4294 NSP3GACACUUUAGAUGAUAUCUUACA  963 CGCACUAAUGGUGGUUACAAUUCUU 132 1 times at4517 NSP3 CGCACUAAUGGUGGUUACAAUUC  964 CCUACUUUCUUACACAGAUUCUAUU 138 1times at 4949 NSP3 UACUUUCUUACACAGAUUCUAUU  965CCGACCUAUCUGCUUUCUAUGUUAA 160 1 times at 5733 NSP3GACCUAUCUGCUUUCUAUGUUAA  966 GGUGAUGCUAUUAGUUUGAGUUUUA 172 1 times at5870 NSP3 GUGAUGCUAUUAGUUUGAGUUUU  967 GCAUCUUAUGAUACUAAUCUUAAUA 176 1times at 6062 NSP3 AUCUUAUGAUACUAAUCUUAAUA  968GCCCCCAUUGAACUCGAAAAUAAAU 178 1 times at 6125 NSP3CCCCAUUGAACUCGAAAAUAAAU  969 CCCCCAUUGAACUCGAAAAUAAAUU 179 1 times at6126 NSP3 CCCAUUGAACUCGAAAAUAAAUU  970 CCUAAGUAUCAAGUCAUUGUCUUAA 184 1times at 6353 NSP3 CCCUAAGUAUCAAGUCAUUGUCU  971GGCUUCAUUUUAUUUCAAAGAAUUU   4 1 times at 6487 NSP3GGCUUCAUUUUAUUUCAAAGAAU  972 CCACUAGCUUACUUUAGUGAUUCAA 194 1 times at6638 NSP3 CACUAGCUUACUUUAGUGAUUCA  973 CCCAAGGUUUGAAAAAGUUCUACAA 203 1times at 6906 NSP3 CCCAAGGUUUGAAAAAGUUCUAC  974CCAAGGUUUGAAAAAGUUCUACAAA 204 1 times at 6907 NSP3AAGGUUUGAAAAAGUUCUACAAA  975 GGCAGGUACAUUGCAUUAUUUCUUU 213 1 times at7207 NSP3 CAGGUACAUUGCAUUAUUUCUUU  976 GCGCUUUUACAAAUCUAGAUAAGUU   5 1times at 7740 NSP3 GCGCUUUUACAAAUCUAGAUAAG  977CGGCUUCAGUUAACCAAAUUGUCUU 242 1 times at 8286 NSP3GGCUUCAGUUAACCAAAUUGUCU  978 CGCAUUGCAUGCCGUAAGUGUAAUU   6 1 times at8387 NSP3 CGCAUUGCAUGCCGUAAGUGUAA  979 CCUCAAAGCUACGCGCUAAUGAUAA 247 1times at 8430 NSP3 CUCAAAGCUACGCGCUAAUGAUA  980CCGCAUCUUGGACUUUAAAGUUCUU 251 1 times at 8638 NSP4CCGCAUCUUGGACUUUAAAGUUC  981 GCUCUUCUAUUAUAUUAAUAAAGUA 279 1 times at9406 NSP4 CUCUUCUAUUAUAUUAAUAAAGU  982 GCUGCCUCUAAUAUCUUUGUUAUUA 285 1times at 9767 NSP4 UGCCUCUAAUAUCUUUGUUAUUA  983CCUCUAAUAUCUUUGUUAUUAACAA 286 1 times at 9771 NSP4CUCUAAUAUCUUUGUUAUUAACA  984 GCAGCUCUUAGAAACUCUUUAACUA 287 1 times at9806 NSP4 CAGCUCUUAGAAACUCUUUAACU  985 CGGAAGUGAAGAUGAUACUUUUAUU 333 1times at 11556 NSP6 CGGAAGUGAAGAUGAUACUUUUA  986GGAAGUGAAGAUGAUACUUUUAUUA 334 1 times at 11557 NSP6AAGUGAAGAUGAUACUUUUAUUA  987 GGCUAUGACUUCUAUGUAUAAGCAA 358 1 times at12259 NSP8 GGCUAUGACUUCUAUGUAUAAGC  988 CCCCAAUCUAAAGAUUCCAAUUUUU 395 1times at 13403 NSP10 CCCCAAUCUAAAGAUUCCAAUUU  990CCCAAUCUAAAGAUUCCAAUUUUUU 396 1 times at 13404 NSP10CCCAAUCUAAAGAUUCCAAUUUU  991 GCUGUGAUGUUACCUACUUUGAAAA 415 1 times at13862 NSP12 CUGUGAUGUUACCUACUUUGAAA  992 CCCAGUGUUAUUGGUGUUUAUCAUA   7 1times at 13915 NSP12 CCCAGUGUUAUUGGUGUUUAUCA  993CCAGUGUUAUUGGUGUUUAUCAUAA 417 1 times at 13916 NSP12CAGUGUUAUUGGUGUUUAUCAUA  994 GGUACAACUCUUUGAGAAGUACUUU 433 1 times at14247 NSP12 UACAACUCUUUGAGAAGUACUUU  995 CCUCCUCUAACGCUUUUCUUGAUUU 446 1times at 14558 NSP12 CUCCUCUAACGCUUUUCUUGAUU  996CCUACUAUGUGUGACAUCAAACAAA 456 1 times at 14791 NSP12UACUAUGUGUGACAUCAAACAAA  997 GCUGGGAUUUCAUGCUUAAAACAUU 475 1 times at15200 NSP12 UGGGAUUUCAUGCUUAAAACAUU  998 GGGAUUUCAUGCUUAAAACAUUGUA   8 1times at 15203 NSP12 GGGAUUUCAUGCUUAAAACAUUG  999CCACUGCAUAUGCCAAUAGUGUCUU 486 1 times at 15467 NSP12CACUGCAUAUGCCAAUAGUGUCU 1000 GGGUGCUAAUGGCAACAAGAUUGUU   9 1 times at15534 NSP12 GGGUGCUAAUGGCAACAAGAUUG 1001 CCCCAAAUUUGUUGAUAAAUACUAU  10 1times at 15624 NSP12 CCCCAAAUUUGUUGAUAAAUACU 1002CGGUUGCUUUGUAGAUGAUAUCGUU  11 1 times at 15930 NSP12CGGUUGCUUUGUAGAUGAUAUCG 1003 GGUUGCUUUGUAGAUGAUAUCGUUA 512 1 times at15931 NSP12 UUGCUUUGUAGAUGAUAUCGUUA 1004 CCCUCUCACAAAGCAUGAAGAUAUA 515 1times at 16011 NSP12 CUCUCACAAAGCAUGAAGAUAUA 1005GGUCUACUUACAGUAUAUAGAAAAA 519 1 times at 16056 NSP12GUCUACUUACAGUAUAUAGAAAA 1006 CCACCACUCAAUCGUAAUUAUGUUU 530 1 times at16726 NSP13 ACCACUCAAUCGUAAUUAUGUUU 1007 CCUACAAGUCUAGUACAACGUAUAA 536 1times at 16835 NSP13 UACAAGUCUAGUACAACGUAUAA 1008GCACUAAUUAUGAUCUUUCAAUUAU 549 1 times at 17342 NSP13CACUAAUUAUGAUCUUUCAAUUA 1009 GCAUGGAGUAAGGCAGUCUUUAUUU 564 1 times at17719 NSP13 AUGGAGUAAGGCAGUCUUUAUUU 1010 GCACAUGCUAACAACAUUAACAGAU 566 1times at 17863 NSP13 CACAUGCUAACAACAUUAACAGA 1011GCCCAAAAAGGUAUUCUUUGUGUUA 568 1 times at 17908 NSP13GCCCAAAAAGGUAUUCUUUGUGU 1012 GCACUCUUUGAGUCCUUAGAGUUUA 571 1 times at17944 NSP13 CACUCUUUGAGUCCUUAGAGUUU 1013 CCUGCGGUUAUGAUUAUGUCUACAA 598 1times at 18689 NSP14 CUGCGGUUAUGAUUAUGUCUACA 1014CGGUUCAUUUGACAAAGUCUAUGAU 607 1 times at 18969 NSP14CGGUUCAUUUGACAAAGUCUAUG 1015 GGUUCAUUUGACAAAGUCUAUGAUA 608 1 times at18970 NSP14 UUCAUUUGACAAAGUCUAUGAUA 1016 GGUAGUAUGAUAGAGGAUAUUGAUU 615 1times at 19360 NSP14 UAGUAUGAUAGAGGAUAUUGAUU 1017GGUGUUAUAAGACCUUUGAUAUUUA 618 1 times at 19517 NSP14GUGUUAUAAGACCUUUGAUAUUU 1018 GGGAUUAUGAACGUAGCAAUAUUUA 629 1 times at19829 NSP15 GGGAUUAUGAACGUAGCAAUAUU 1019 GCCAUCUUUAUUUCUGAUAGAAAAA 634 1times at 19972 NSP15 GCCAUCUUUAUUUCUGAUAGAAA 1020CCAUCUUUAUUUCUGAUAGAAAAAU 635 1 times at 19973 NSP15AUCUUUAUUUCUGAUAGAAAAAU 1021 CCGUGAUAGUGAUGUUGUUAAACAA 637 1 times at20055 NSP15 CCGUGAUAGUGAUGUUGUUAAAC 1022 GGUCUUCACUUGCUUAUUGGUUUAU 643 1times at 20302 NSP15 GUCUUCACUUGCUUAUUGGUUUA 1023GCUCAACUAUUCAUAACUAUUUUAU 648 1 times at 20378 NSP15CUCAACUAUUCAUAACUAUUUUA 1024 GGUUCCUAUUGACUUAACAAUGAUU 657 1 times at20520 NSP15 UUCCUAUUGACUUAACAAUGAUU 1025 CCCUCUUUAAAGUUCAAAAUGUAAA 661 1times at 20639 NSP16 CUCUUUAAAGUUCAAAAUGUAAA 1026CCUGCCAAUAUGCGUGUUAUACAUU 669 1 times at 20785 NSP16UGCCAAUAUGCGUGUUAUACAUU 1027 CCGACAUGUAUGAUCCUACUACUAA 675 1 times at20987 NSP16 GACAUGUAUGAUCCUACUACUAA 1028 GGAGCGUUGAACUUUAUGAACUUAU 682 1times at 21128 NSP16 GAGCGUUGAACUUUAUGAACUUA 1029GGGUACUAUUAAAGAAAAUAUAGAU 687 1 times at 21237 NSP16GGGUACUAUUAAAGAAAAUAUAG 1030 GGCCGUACAUAUUCUAACAUAACUA  12 1 times at21635 S protein GGCCGUACAUAUUCUAACAUAAC 1031 GCCGUACAUAUUCUAACAUAACUAU700 1 times at 21636 S protein GCCGUACAUAUUCUAACAUAACU 1032GGGAGACCAUGGUGAUAUGUAUGUU 703 1 times at 21688 S proteinCACUUUACUUAGAGCUUUUUAUU 1033 GCACCUUUAUGUACACUUAUAACAU 717 1 times at22164 S protein CACCUUUAUGUACACUUAUAACA 1034 CCGAAGAUGAGAUUUUAGAGUGGUU 13 1 times at 22191 S protein CCGAAGAUGAGAUUUUAGAGUGG 1035GGUCCAAUAUCCCAGUUUAAUUAUA 741 1 times at 22838 S proteinUGGUCCAAUAUCCCAGUUUAAUU 1036 CCCAGUUUAAUUAUAAACAGUCCUU  14 1 times at22848 S protein CCCAGUUUAAUUAUAAACAGUCC 1037 GGUUGAUCAACUUAAUAGUAGUUAU765 1 times at 23761 S protein UUGAUCAACUUAAUAGUAGUUAU 1038GCCAGGAUGAUUCUGUACGUAAUUU 770 1 times at 23976 S proteinCAGGAUGAUUCUGUACGUAAUUU 1039 GGAUGAUUCUGUACGUAAUUUGUUU 771 1 times at23980 S protein AUGAUUCUGUACGUAAUUUGUUU 1040 CCAGGUUUUGGAGGUGACUUUAAUU774 1 times at 24041 S protein CAGGUUUUGGAGGUGACUUUAAU 1041GGCUUCACUACAACUAAUGAAGCUU  15 1 times at 24485 S proteinGGCUUCACUACAACUAAUGAAGC 1042 CCCCUGUUAAUGGCUACUUUAUUAA  16 1 times at24951 S protein CCCCUGUUAAUGGCUACUUUAUU 1043 CCCUGUUAAUGGCUACUUUAUUAAA 17 1 times at 24952 S protein CCCUGUUAAUGGCUACUUUAUUA 1044GCACAAACUGUAUGGGAAAACUUAA 813 1 times at 25425 S proteinCACAAACUGUAUGGGAAAACUUA 1045 GCCGCAUAAGGUUCAUGUUCACUAA  18 1 times at25492 S protein GCCGCAUAAGGUUCAUGUUCACU 1046 GCUGCGCAAAACUCUUGUUCUUAAU841 1 times at 26481 orf4b CUGCGCAAAACUCUUGUUCUUAA 1047CGCAAAACUCUUGUUCUUAAUGCAU 842 1 times at 26485 orf4bCGCAAAACUCUUGUUCUUAAUGC 1048 GGCUUUCUCGGCGUCUUUAUUUAAA 852 1 times at26841 orf5 GGCUUUCUCGGCGUCUUUAUUUA 1049 CCUUGUUCUGUAUAACUUUUUAUUA 854 1times at 27057 orf5 UUGUUCUGUAUAACUUUUUAUUA 1050GGACAUAUGGAAAACGAACUAUGUU 873 1 times at 27569 GACAUAUGGAAAACGAACUAUGU1051 GGCAUUGUAGCAGCUGUUUCAGCUA 884 1 times at 28092 MGGCAUUGUAGCAGCUGUUUCAGC 1052 GCCUAUUACGGCGGAUAUUGAACUU 903 1 times at28469 M GCCUAUUACGGCGGAUAUUGAAC 1053 CCGGUACUAAGCUUCCUAAAAACUU 923 1times at 29019 N CCGGUACUAAGCUUCCUAAAAAC 1054

TABLE 3 Predicted 25 mer siRNA targeting 25mer blunt ended sequencesSEQ ID Start Protein 23 mer Sequences passing all SEQ ID SiRNA sequenceNO: Base Name metrics and BLAST search NO: CCCAGAAUCUGCUUAAGAAGUUGAU  321 times at 825 NSP1 CCCAGAAUCUGCUUAAGAAGUUG  952GCCCAUUCAUGGAUAAUGCUAUUAA  64 1 times at 1884 NSP2GCCCAUUCAUGGAUAAUGCUAUU  953 CCCAUUCAUGGAUAAUGCUAUUAAU  65 1 times at1885 NSP2 CCCAUUCAUGGAUAAUGCUAUUA  954 CGCCAUUACUGCACCUUAUGUAGUU  67 1times at 1936 NSP2 CGCCAUUACUGCACCUUAUGUAG  955GGCGACUUUAUGUCUACAAUUAUUA  71 1 times at 2186 NSP2GGCGACUUUAUGUCUACAAUUAU  957 CGCAAUACGUAAAGCUAAAGAUUAU   1 1 times at4144 NSP3 CGCAAUACGUAAAGCUAAAGAUU  961 GGGUGUUGAUUAUACUAAGAAGUUU   2 1times at 4228 NSP3 GGGUGUUGAUUAUACUAAGAAGU  962CGCACUAAUGGUGGUUACAAUUCUU 132 1 times at 4517 NSP3CGCACUAAUGGUGGUUACAAUUC  964 GGCUUCAUUUUAUUUCAAAGAAUUU   4 1 times at6487 NSP3 GGCUUCAUUUUAUUUCAAAGAAU  972 GCGCUUUUACAAAUCUAGAUAAGUU   5 1times at 7740 NSP3 GCGCUUUUACAAAUCUAGAUAAG  977CGCAUUGCAUGCCGUAAGUGUAAUU   6 1 times at 8387 NSP3CGCAUUGCAUGCCGUAAGUGUAA  979 CCGCAUCUUGGACUUUAAAGUUCUU 251 1 times at8638 NSP4 CCGCAUCUUGGACUUUAAAGUUC  981 CGGAAGUGAAGAUGAUACUUUUAUU 333 1times at 11556 NSP6 CGGAAGUGAAGAUGAUACUUUUA  986GGCUAUGACUUCUAUGUAUAAGCAA 358 1 times at 12259 NSP8GGCUAUGACUUCUAUGUAUAAGC  988 CCCCAAUCUAAAGAUUCCAAUUUUU 395 1 times at13403 NSP10 CCCCAAUCUAAAGAUUCCAAUUU  990 CCCAAUCUAAAGAUUCCAAUUUUUU 936 1times at 13404 NSP10 CCCAAUCUAAAGAUUCCAAUUUU  991CCCAGUGUUAUUGGUGUUUAUCAUA   7 1 times at 13915 NSP12CCCAGUGUUAUUGGUGUUUAUCA  993 GGGAUUUCAUGCUUAAAACAUUGUA   8 1 times at15203 NSP12 GGGAUUUCAUGCUUAAAACAUUG  999 GGGUGCUAAUGGCAACAAGAUUGUU   9 1times at 15534 NSP12 GGGUGCUAAUGGCAACAAGAUUG 1001CCCCAAAUUUGUUGAUAAAUACUAU  10 1 times at 15624 NSP12CCCCAAAUUUGUUGAUAAAUACU 1002 CGGUUGCUUUGUAGAUGAUAUCGUU  11 1 times at15930 NSP12 CGGUUGCUUUGUAGAUGAUAUCG 1003 GCCCAAAAAGGUAUUCUUUGUGUUA 568 1times at 17908 NSP13 GCCCAAAAAGGUAUUCUUUGUGU 1012CGGUUCAUUUGACAAAGUCUAUGAU 607 1 times at 18969 NSP14CGGUUCAUUUGACAAAGUCUAUG 1015 GGGAUUAUGAACGUAGCAAUAUUUA 629 1 times at19829 NSP15 GGGAUUAUGAACGUAGCAAUAUU 1019 GCCAUCUUUAUUUCUGAUAGAAAAA 634 1times at 19972 NSP15 GCCAUCUUUAUUUCUGAUAGAAA 1020CCGUGAUAGUGAUGUUGUUAAACAA 637 1 times at 20055 NSP15CCGUGAUAGUGAUGUUGUUAAAC 1022 GGGUACUAUUAAAGAAAAUAUAGAU 687 1 times at21237 NSP16 GGGUACUAUUAAAGAAAAUAUAG 1030 GGCCGUACAUAUUCUAACAUAACUA  12 1times at 21635 S protein GGCCGUACAUAUUCUAACAUAAC 1031GCCGUACAUAUUCUAACAUAACUAU 700 1 times at 21636 S proteinGCCGUACAUAUUCUAACAUAACU 1032 CCGAAGAUGAGAUUUUAGAGUGGUU  13 1 times at22191 S protein CCGAAGAUGAGAUUUUAGAGUGG 1035 CCCAGUUUAAUUAUAAACAGUCCUU 14 1 times at 22848 S protein CCCAGUUUAAUUAUAAACAGUCC 1037GGCUUCACUACAACUAAUGAAGCUU  15 1 times at 24485 S proteinGGCUUCACUACAACUAAUGAAGC 1042 CCCCUGUUAAUGGCUACUUUAUUAA  16 1 times at24951 S protein CCCCUGUUAAUGGCUACUUUAUU 1043 CCCUGUUAAUGGCUACUUUAUUAAA 17 1 times at 24952 S protein CCCUGUUAAUGGCUACUUUAUUA 1044GCCGCAUAAGGUUCAUGUUCACUAA  18 1 times at 25492 S proteinGCCGCAUAAGGUUCAUGUUCACU 1046 CGCAAAACUCUUGUUCUUAAUGCAU 842 1 times at26485 orf4b CGCAAAACUCUUGUUCUUAAUGC 1048 GGCUUUCUCGGCGUCUUUAUUUAAA 852 1times at 26841 orf5 GGCUUUCUCGGCGUCUUUAUUUA 1049GGCAUUGUAGCAGCUGUUUCAGCUA 884 1 times at 28092 M GGCAUUGUAGCAGCUGUUUCAGC1052 GCCUAUUACGGCGGAUAUUGAACUU 903 1 times at 28469 MGCCUAUUACGGCGGAUAUUGAAC 1053 CCGGUACUAAGCUUCCUAAAAACUU 923 1 times at29019 N CCGGUACUAAGCUUCCUAAAAAC 1054

TABLE 4 Characterization indexes of five SLiC species and fiveSLiC-siRNA nanoparticles, including particle sizes, poly-dispersityindex (PDI) and Zeta-potential. Names Diameter (nm) PDI Zeta-potential(mV0) SLiC1 479.3 ± 55.1 0.66 ± 0.13 61.1 ± 1.27 SLiC2 196.9 ± 25.6 0.41± 0.24 42.3 ± 1.85 SLiC3 213.8 ± 20.4 0.25 ± 0.14 43.1 ± 1.72 SLiC4341.2 ± 33.8 0.71 ± 0.08 46.1 ± 1.35 SLiC5  1091 ± 34.2 0.87 ± 0.09 61.5± 1.14 SLiC1 (siRNA) 174.1 ± 11.1 0.39 ± 0.03 42.3 ± 1.15 SLiC2 (siRNA)115.5 ± 15.6 0.20 ± 0.04 34.4 ± 1.85 SLiC3 (siRNA) 169.6 ± 10.4 0.22 ±0.04 38.2 ± 0.80 SLiC4 (siRNA) 154.7 ± 13.8 0.35 ± 0.07 40.6 ± 1.21SLiC5 (siRNA) 182.6 ± 14.1 0.38 ± 0.09 44.4 ± 1.23

What is claimed is:
 1. A pharmaceutical composition comprising at leasttwo different siRNA molecules that target the MERS-CoV Spike protein,and a pharmaceutically acceptable carrier comprising a polymericnanoparticle or a liposomal nanoparticle, wherein the siRNA moleculesare selected from the group consisting of: (SEQ ID NO: 12)MSP1: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 700)MSP2: GCCGUACAUAUUCUAACAUAACUAU,  (SEQ ID NO: 13)MSP3: CCGAAGAUGAGAUUUUAGAGUGGUU,  (SEQ ID NO: 14)MSP4: CCCAGUUUAAUUAUAAACAGUCCUU,  (SEQ ID NO: 15)MSP5: GGCUUCACUACAACUAAUGAAGCUU,  (SEQ ID NO: 16)MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,  (SEQ ID NO: 17)MSP7: CCCUGUUAAUGGCUACUUUAUUAAA,  and (SEQ ID NO: 18)MSP8: GCCGCAUAAGGUUCAUGUUCACUAA. 


2. A pharmaceutical composition comprising at least two different siRNAmolecules that target the genome of a MERS-CoV, wherein a first siRNAmolecule comprises MRR2: GGGAUUUCAUGCUUAAAACAUUGUA (SEQ ID NO: 20) and asecond siRNA molecule comprises MSP2: GCCGUACAUAUUCUAACAUAACUAU (SEQ IDNO: 700).
 3. A pharmaceutical composition comprising a siRNA cocktail,MSTRS1, wherein a first siRNA molecule comprises MRR1:CCCAGUGUUAUUGGUGUUUAUCAUA (SEQ ID NO: 21) and a second siRNA moleculecomprises MSP1: GGCCGUACAUAUUCUAACAUAACUA (SEQ ID NO: 12) and apharmaceutically acceptable carrier comprising a polymeric nanoparticleor a liposomal nanoparticle.
 4. A pharmaceutical composition comprisinga siRNA cocktail, MSTPRS1, wherein a first siRNA molecule comprisesMPL1: CGCAAUACGUAAAGCUAAAGAUAU (SEQ ID NO: 22), a second siRNA moleculecomprises MRR1: CCCAGUGUUAUUGGUGUUUAUCAUA (SEQ ID NO: 7), and a thirdsiRNA molecule comprises MSP1: GGCCGUACAUAUUCUAACAUAACUA (SEQ ID NO:12)and a pharmaceutically acceptable carrier comprising a polymericnanoparticle or a liposomal nanoparticle.
 5. The composition of claim 1,wherein the polymeric nanoparticle carrier comprises a Histidine-Lysineco-polymer (HKP).
 6. The composition of claim 1, wherein the liposomalnanoparticle carrier comprises a Spermine-Lipid Conjugate (SLiC) andcholesterol.
 7. A method of treating a mammal with a MERS infectioncomprising administering to said mammal a pharmaceutically effectiveamount of the composition of claim
 1. 8. The method of claim 7, whereinthe mammal is a human.
 9. An siRNA molecule that targets a conservedregion of the genome of a MERS-CoV wherein the molecule is selected fromthe group consisting of the molecules of SEQ ID NOs 12, 1031, 700, 1032,703, 1033, 717, 1034, 13, 1035, 741, 1036, 14, 1037, 765, 1038, 770,1039, 771, 1040, 774, 1041, 15, 1042, 16, 1043, 17, 1044, 813, 1045, 18,and
 1046. 10. An siRNA molecule that targets a conserved region of thegenome of a MERS-CoV selected from the group consisting of: (SEQ ID NO: 12) MSP1: GGCCGUACAUAUUCUAACAUAACUA, (SEQ ID NO: 700)MSP2: GCCGUACAUAUUCUAACAUAACUAU,  (SEQ ID NO: 13)MSP3: CCGAAGAUGAGAUUUUAGAGUGGUU,  (SEQ ID NO: 14)MSP4: CCCAGUUUAAUUAUAAACAGUCCUU,  (SEQ ID NO: 15)MSP5: GGCUUCACUACAACUAAUGAAGCUU,  (SEQ ID NO: 16)MSP6: CCCCUGUUAAUGGCUACUUUAUUAA,  (SEQ ID NO: 17)MSP7: CCCUGUUAAUGGCUACUUUAUUAAA,  and  (SEQ ID NO: 18)MSP8: GCCGCAUAAGGUUCAUGUUCACUAA.


11. A composition comprising the siRNA molecule of claim 9 and apharmaceutically acceptable carrier comprising a polymeric nanoparticleor a liposomal nanoparticle.
 12. A method of treating a mammal with aMERS infection comprising administering to said mammal apharmaceutically effective amount of the composition of claim
 11. 13.The method of claim 12, wherein the mammal is a human.
 14. Thecomposition of claim 2, wherein if a polymeric nanoparticle carrier ispresent said carrier comprises a Histidine-Lysine co-polymer (HKP) andif a liposomal nanoparticle carrier is present said carrier comprises aSpermine-Lipid Conjugate (SLiC) and cholesterol.
 15. The composition ofclaim 3, wherein if a polymeric nanoparticle carrier is present saidcarrier comprises a Histidine-Lysine co-polymer (HKP) and if a liposomalnanoparticle carrier is present said carrier comprises a Spermine-LipidConjugate (SLiC) and cholesterol.
 16. The composition of claim 4,wherein if a polymeric nanoparticle carrier is present said carriercomprises a Histidine-Lysine co-polymer (HKP) and if a liposomalnanoparticle carrier is present said carrier comprises a Spermine-LipidConjugate (SLiC) and cholesterol.
 17. A method of treating a mammal witha MERS infection comprising administering to said mammal apharmaceutically effective amount of the composition of claim
 2. 18. Amethod of treating a mammal with a MERS infection comprisingadministering to said mammal a pharmaceutically effective amount of thecomposition of claim
 3. 19. A method of treating a mammal with a MERSinfection comprising administering to said mammal a pharmaceuticallyeffective amount of the composition of claim
 4. 20. A method of treatinga mammal with a MERS infection comprising administering to said mammal apharmaceutically effective amount of the composition of claim 10.